s2fieldsofstudy: Biology Medicine

text:
Modulation of soluble guanylate cyclase ameliorates pulmonary hypertension in a rat model of chronic thromboembolic pulmonary hypertension by stimulating angiogenesis

Abstract Acute pulmonary embolism (PE) does not always resolve after treatment and can progress to chronic thromboembolic disease (CTED) or the more severe chronic thromboembolic pulmonary hypertension (CTEPH). The mechanisms surrounding the likelihood of PE resolution or progress to CTED/CTEPH remain largely unknown. We have developed a rat model of CTEPH that closely resembles the human disease in terms of hemodynamics and cardiac manifestations. Embolization of rats with polystyrene microspheres followed by suppression of angiogenesis with the inhibitor of vascular endothelial growth factor receptor 2 (VEGF‐R2) SU5416 results in transient, acute pulmonary hypertension that progresses into chronic PE with PH with sustained right ventricular systolic pressures exceeding 70 mmHg (chronic pulmonary embolism [CPE] model). This model is similar to the widely utilized hypoxia/SU5416 model with the exception that the “first hit” is PE. Rats with CPE have impaired right heart function characterized by reduced VO2Max, reduced cardiac output, and increased Fulton index. None of these metrics are adversely affected by PE alone. Contrast‐mediated CT imaging of lungs from rats with PE minus SU5416 show large increases in pulmonary vascular volume, presumably due to an angiogenic response to acute PE/PH. Co‐treatment with SU5416 suppresses angiogenesis and produces the CTEPH‐like phenotype. We report here that treatment of CPE rats with agonists for soluble guanylate cyclase, a source of cGMP which is in turn a signal for angiogenesis, markedly increases angiogenesis in lungs, and ameliorates the cardiac deficiencies in the CPE model. These results have implications for future development of therapies for human CTEPH.

| INTRODUCTION
Each year, approximately 600,000 persons in the US have PE diagnosed, and 30,000 die, making PE the third most common cause of cardiovascular death in the US (AHRQ Quality, 2018; Barco et al., 2020;Mozaffarian et al., 2015). The 30-day case fatality rate for PE remains approximately 6%-8% overall and for severe PE (any arterial hypotension), the case fatality rate increases to 35% (Alotaibi et al., 2016;Spencer et al., 2010;Stein et al., 2012). Patients with PE that causes acute PH are at higher risk of RV failure and death within a few days (Jaff et al., 2011;Konstantinides et al., 2014). Treatment options for patients with more severe PE who are more likely to progress to PH remain limited to the acute use of systemic or catheter-delivered fibrinolytic agents, which increase bleeding risk without a clear mortality improvement or benefit to RV function (Chatterjee et al., 2014;Nakamura et al., 2014;Riera-Mestre et al., 2014;Tafur et al., 2017).
About 30% of patients with PE go on to have chronic thromboembolic disease (CTED), which in turn manifests a clinical spectrum, ranging from mild exercise-induced fatigue and dyspnea (post-PE syndrome) (Klok, van der Hulle, et al., 2016) to devastating chronic thromboembolic pulmonary hypertension (CTEPH) (Fernandes et al., 2016;Held et al., 2016;Kline et al., 2017;Sista et al., 2017). CTED appears to result from a combination of factors, including hypofibrinolysis and recurrent PE (Lang et al., 2008;Fernandes et al., 2016). Although understanding of the progression of CTED is poor, current data suggest about 10% of patients have progressive PH and develop the full blown CTEPH syndrome, defined by persistent perfusion defects and a resting mPAP>35 mmHg with LVEDP <15 mmHg (Nijkeuter et al., 2007;Pepke-Zaba et al., 2011). At present, the only FDA-approved drug to treat CTEPH is the soluble guanylate cyclase (sGC) stimulator, riociguat (RIO) (Dasgupta et al., 2015). Currently, RIO is administered after CTEPH diagnosis and is thought to work by causing pulmonary arterial smooth muscle relaxation.
It is unclear why only a fraction of patients with equal degree of pulmonary vascular obstruction from PE go on to develop PH with variable severity. The answer is not simply the degree of clot blockage or its persistence (Es et al., 2013;Exter et al., 2015;Klok, Dzikowska-Diduch, et al., 2016;Morris, 2013). Development of PH requires blockage plus vasospasm and maladaptive remodeling, resulting in permanent decrease in cross-sectional vascular area (Delcroix et al., 2016;Gopalan et al., 2017). These findings have raised the "misguided angiogenesis" hypothesis as a mechanism of CTEPH, and conversely, increased angiogenesis can prevent CTEPH by two broad mechanisms (Alias et al., 2014;Quarck et al., 2015). First, new vessels increase cross-sectional vascular area thus decreasing pulmonary vascular resistance, and second by enhanced vascular ingrowth into thrombi hastening their resolution (Alias et al., 2014;Sharma & Lang, 2018). Angiogenesis refers to the sprouting of new vessels from existing vessels, driven by extension, and proliferation of endothelial cells. Vascular endothelial growth factor (VEGF) is the master regulator of both the growth and maintenance of pulmonary vessels, although VEGF-driven angiogenesis is enhanced by, and may even require, other growth factors, notably PDGF (Heldin et al., 1998;Hellberg et al., 2010;Voelkel & Gomez-Arroyo, 2014). Disruption of VEGF signaling with the VEGF receptor 2-selective tyrosine kinase inhibitor SU5416 is well known to convert hypoxia, which alone seldom produces PH, into an animal model with a nearly 100% rate of PH (Voelkel & Gomez-Arroyo, 2014). sGC is a heterodimeric protein which is the receptor for the important vasodilator nitric oxide (NO; 34). Binding of NO to sGC requires the presence of a heme moiety with a coordinated ferrous iron ion, Fe 2+ (Derbyshire & Marletta, 2012). Oxidation of ferrous iron to ferric iron Fe 3+ results in loss of the heme moiety, non-responsiveness to NO and impaired vasodilation. Pharmacologic agonists have been developed to modulate sGC activity (Brusilovskaya et al., 2019;Priviero & Webb, 2010;Schermuly et al., 2011). These compounds behave by two distinct mechanisms. sGC stimulators bind to and are agonists for native hemecontaining, ferrous sGC (example, riociguat, RIO, BAY 63-2521) while sGC activators bind to and are agonists for heme-free sGC lacking its iron ion (example, cinaciguat, CINA, BAY 58-2667;11;Kollau et al., 2018). sGC stimulators sensitize the reduced form of sGC to endogenous and exogenous NO. On the other hand, sGC activators re-activate oxidized or heme-free states of sGC that have become unresponsive to NO due to disease processes involving increased oxidative stress (Evgenov et al., 2006).
We have previously described a novel rat model of chronic pulmonary embolism (CPE) that combines embolization with polystyrene microspheres and suppression of angiogenesis by post-PE treatment with SU5416 (Neto-Neves et al., 2017). Rats embolized with microspheres in the absence of SU5416 rarely develop chronic, progressive PH. Barium-contrasted lung CT scans of these rats reveal extensive new pulmonary vessel growth 3-4 weeks post-PE and a 75% increase in vascular bed volume essentially rescuing the lungs from the effects of the embolization. Significantly, when embolized rats are administered a single dose of SU5416, rats fail to show pulmonary angiogenesis by barium-contrast CT and 95% develop severe PH, effectively modelling the human CTEPH phenotype (Neto-Neves et al., 2017). In this current study we report on the effects of the sGC agonists RIO and CINA on pulmonary angiogenesis and reduction in chronic PH.

| Rats
Weighted 350 to 380-grams male Sprague-Dawley rats were purchased from Harlan Sprague Dawley. All rats had free access to food and water prior to use.

| Chronic pulmonary embolism model
CPE was induced in rats as previously described by our lab with some modifications (Neto-Neves et al., 2017). Briefly, rats were sedated with isoflurane/O 2 under controlled inhalation conditions and PE induced by injection in the tail vein with 90 μl/100g body weight of 85-micron copolymer microspheres (Thermo Fisher #184942). This dose was empirically determined to produce RVSP of ≥50 mmHg by catheterization of the right jugular vein using 2 French Millar pressure transducing catheters (Millar-AD Instruments NA; Colorado Springs CO # SPR-513) and the LabView2013 Pressure Precision T1600vi biometric software (National Instruments). One day after injection of microspheres rats were injected subcutaneously with 1ml/kg of SU5416 (Tocris 3037 or MedChem Express HY-10374) dissolved in DMSO at 20 mg/ml (20 mg/kg final dose). Treatment of rats with CINA or RIO began at 7 days post-PE for a total of 2 weeks. CINA (Tocris 6052 or BAY 582667; gifted from Bayer-AG) or RIO (MedChem Express, #HY-14779) was administered to rats by subcutaneous injection twice daily with 0.83 ml/ kg chemical dissolved in DMSO at 3 mg/ml (2.5 mg/kg/ injection final dose). Control rats received a fixed dose of DMSO at 0.3 ml/injection.

| Terminal measurements and tissue harvests
Rats were anesthetized with continuous isoflurane inhalation. RVP measurements were made as described above. Measurements of VO 2 Max and cardiac output have been previously described , Watts et al., 2006. Whole blood was withdrawn into EDTAcontaining tubes and plasma separated by high-speed centrifugation (1 min at 10,000 g). Lungs were extensively perfused with saline solution by cannulating the pulmonary artery with a 25-gauge syringe needle. The left lung lobe was transferred into 10% neutral buffered formalin for fixation and the right lobes were freeze-clamped and stored at −80°C. Hearts were excised and separated into RV and LV+septum sections, weighed for Fulton index metrics and then freeze-clamped and stored at −80°C. In some rats, the thoracic descending aorta and pulmonary artery (main PA plus left and right branches) were carefully dissected and placed in ice-cold saline for subsequent processing into aortic and PA rings.

| Lung vascular volume
Lung vascular volume was quantified by high resolution compute tomography as previously described (Neto-Neves et al., 2017). After euthanasia, heparinized saline (99-parts 0.9% NaCl and 1-part Heparin) was infused through the right jugular vein to flush the pulmonary circulation free of blood. A barium sulfate contrast media (BriteVu; Scarlett imaging LLC) was infused through the jugular vein catheter with the lungs inflated with air (20 mmHg) and trachea was ligated to maintain pressure. Heart and lung blocks were excised and placed on a carbon-fiber bed and scanned with small animal high resolution micro-CT scanner (Skyscan 1176; Bruker Biospin Corp). The micro-CT images were acquired at 33.63 μm using three continuous bed positions to cover the entirety of the lungs, where the tube voltage, current, step-size filter, and exposure times will be 80 kV, 313 μA, 0.9 degree/step, 0.5 mm Al filter, and 20 ms, respectively. Upon completion of the micro-CT scans, images were reconstructed using filtered back projection via SkyScan NRecon software (version 1.6.9.4), where a Hamming filter of 0.25 (alpha) and Nyquist frequency cutoff of 100 Hz yielded an isotropic voxels with a 33.63 μm resolution. For analysis, DICOM image volumes will be imported into Analyze 12.0 (AnalyzeDirect), signal intensity normalized (i.e., [0.0, 1.0]), and tissue segmentation accomplished via segmented threshold for background+soft tissue (i.e., 0.0-0.1) and lung vasculature (i.e., 0.1-1.0). Segmented object maps are then quantified for total vascular volumes using Analyze's ROITool.

| Aortic and pulmonary artery ring assays in Geltrex
Aortic rings were dissected almost exclusively from the aortic arch and adjacent descending thoracic aorta but in some hearts it was necessary to cut rings from the ascending aorta. Pulmonary artery rings were preferentially dissected from the left (narrower) pulmonary artery, but it was often necessary to also cut rings from the right (much wider) pulmonary artery along with left rings in the same experiment. A 96-well cell culture plates were prepared for ring assays by applying 20 μL of cold Geltrex artificial basement membrane (Gibco-Thermo Fisher #A1413202) per well to plates kept on ice and then centrifuging the plates for 10 min at 300 rpm and 4°C to distribute the Geltrex and eliminate air bubbles. Plates were stored at 37°C for at least 30 min to allow for gelling. Plates were then put on ice again and an additional 40 μl of Geltrex applied over the previous 20 μl. Rings were carefully cut in sterile plastic dishes containing sterile serum-free rat endothelial cell medium (Cell Biologics, M1266) removed from dissection dishes with sterile forceps, dipped into for M1266 for cleaning, dried very carefully with sterile gauze and inserted into the ice-cold liquid Geltrex in wells. After all rings had been placed, plates were stored at 37°C for at least 30 min for gelling. Geltrex-embedded rings were treated with 100 μl of M1266 media containing appropriate additives, which was changed daily. At the end of 10 days rings were labeled with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to enhance visibility of sprouts then fixed with formaldehyde. Rings were imaged at 4x magnification using an Evos phase contrast microscope.

| Endothelial tube assay on Geltrex
Rat pulmonary microvascular endothelial cells (PMVEC; Cell Biologics RA6011) or pulmonary artery endothelial cells (PAEC; Cell Biologics RA6059) were grown in complete M1266 medium until approximately ¼ to ½ confluent, washed with PBS and starved overnight in basal M1266 plus 0.1% serum but lacking supplemental growth factors (EGF, VEGF). Sub-confluent cells were harvested with TrypLE trypsin (Gibco Thermo Fisher #12605093), diluted with basal M1266 basal medium (serum-free, growth factor-free), strained through 40-micron cell strainers (Sigma #CLS 431750) to eliminate aggregates and collected by centrifugation. Tube assays were done in 96 well plates containing 30 μl/well pre-aliquoted and gelled Geltrex. Aliquots (100 μl) of cells PMVECs, 25k/ well; PAECs, 20k/well) plus supplements were aliquoted on to Geltrex and tubes allowed to form overnight. Tubes were then fixed with 10% neutral buffered formalin and imaged by phase contrast microscopy. All images were enhanced equally in Image J with the following commands: Image>Adjust>Brightness/Contrast>Auto; Process>Smooth; Process>Noise>Despeckle. Angiogenic metrics of tubes were measured with the Angiogenesis Analyzer plug-in for Image J software using default settings.

| Study approval
All animal experiments were approved by the IACUC at Indiana University-Purdue University Indianapolis (IUPUI), Indiana University School of Medicine, Indianapolis. The approved protocol number was 11162 assigned to Jeffrey A. Kline, MD as Principal Investigator.

| CINA reduces right ventricular systolic pressure and RV damage in rats with PH caused by CPE
Dosing of rats with SU5416 following microsphere PE resulted in elevated right ventricular systolic pressure (RVSP) at 28 days post-PE. Treatment of CPE rats with CINA or RIO reduced RVSP to near normal values (Figure 1a), accompanied by proportional improvements in reduced VO 2 Max (1b), cardiac output (1c), and Fulton index (mass RV/mass LV+septum) (1d). Telemetric monitoring of rat RV and aortic pressures over a 7-week time frame revealed PH development with PE+SU5416 (Figure 2 top). RVSP spiked briefly following microsphere infusion, consistent with our previously published results using an earlier PE model (Watts et al., 2006(Watts et al., , 2008Zagorski et al., 2003Zagorski et al., , 2007. RVSP increased linearly for approximately 3 weeks and then remained elevated for 7 weeks post-PE. Left ventricular pressure was not significantly altered. Delayed administration of CINA to CPE rats reversed the acute rise in RVSP following microsphere infusion (Figure 2 bottom, days 0-13) and normalized RVSP during the entire 7-week monitoring period even after discontinuation of CINA dosing at day 20. Curiously, LVSP increased near the end of the CINA treatment period and continued to increase until reaching a variable plateau of ~130-150 mm pressure. Telemetric monitoring was not done with RIO-treated rats.

| CINA or RIO treatment rescues pulmonary angiogenesis
As previously reported, rats with CPE have increased pulmonary vascular volume as determined by post-mortem barium-contrast CT imagery followed by 3-D reconstruction for volume determination, while rats also receiving SU5416 had greatly reduced angiogenesis (Neto-Neves et al., 2017). The addition of a 2-week course of CINA or RIO treatment to CPE rats resulted in significantly increased pulmonary vascular volume (Figure 3 top). This effect was evident in CT imaging of barium-contrasted lungs in rats treated with CINA (Figure 3d vs. 3c). Despite the ability to quantify volume, because of technical problems with the 2-D to 3-D image assembly process, we were not able to obtain high quality images of lungs from RIO-treated rats. Taken with the salutary effect of CINA on RV function, these data implied a protective effect of increased angiogenesis following PE which was inhibited by SU5416 and rescued by activation/stimulation of sGC by CINA and RIO.
To better understand the function of these new vessels in gas exchange, in a subset of anesthetized, mechanically ventilated animals, we obtained arterial blood via femoral artery cut down and cannulation, and measured partial pressures of oxygen and carbon dioxide, while simultaneously measuring the exhaled end-tidal CO 2 . These measurements allowed calculation of the arterial-alveolar oxygen gradient (arterial PO 2 -alveolar CO 2 ) and alveolar dead space estimate from the Severinghaus equation ([PaCO 2 -PetCO 2 ]/PaCO 2 ). These data in Figure 4 suggested an increase in shunt fraction without an increase in alveolar dead space, again supporting the hypothesis of intrapulmonary vascularization out of proportion to alveolar volume. Furthermore, the bronchoalveolar lavage fluid (BALF) concentration of VEGfA (measured by enzymelinked immunoassay) was significantly elevated in PE rats versus controls using ELISA measurements ( Figure 5).

| Aortic and PA explants retain their angiogenic phenotypes when cultured as rings in Geltrex basement membrane preparation
Aortas and pulmonary arteries were dissected from CPE rats treated with either DMSO (control), CINA, or RIO F I G U R E 1 Plot of in vivo values obtained from rats. Right ventricular systolic pressure (RVSP) was measured with a micromanometer inserted via the jugular vein in anesthetized rats. VO 2 Max was the total oxygen consumption measured at peak exercise; cardiac output was measured in lightly anesthetized rats with echocardiography; the Fulton index (mass RV/[mass septum+LV]) was obtained at terminal harvest. CINA, cinaciguate; PE, pulmonary embolism; RIO, riociguate; SU, SU5416. The asterisk indicates p < 0.05 by Tukey's post hoc following one-way ANOVA versus all other groups. Sample sizes are represented by dots in the figure and assayed for angiogenic phenotype using Geltrex ring assays. These data are shown in Figure 6 for aortic (top panels) and PA rings (bottom panels). Images are shown for rings which were metabolically labeled with MTT to enhance the visibility of otherwise faint angiogenic sprouts. Rings obtained from CPE rats treated with either CINA or RIO had more sprout formation than rings from control rats receiving only DMSO. Aortic rings from rats treated with CINA showed the largest increase in sprout formation relative to controls. These ex vivo data were consistent with the enhanced vascularization of pulmonary vessels seen in vivo by barium-enhanced CT. The data also indicate that the angiogenic phenotype of vessels exposed to CINA and RIO in rats was maintained for at least 1-week post treatment in vivo and that the phenotype was maintained through at least 10 days of ex vivo culture.

F I G U R E 2
Aortic and right ventricular pressures measured over 50 days in two groups of conscious, freely moving rats, instrumented with telemetrically connected micromanometers. The top panel shows the effect of PE+SU5416 (marked by PE in box) without further treatment (n = 3). The bottom panel shows the effect of PE+SU5416, followed 7 days later by 10 mg/kg/day of cinaciguat, injected subcutaneously daily (n = 3). The data show the mean of 1 min of monitoring, taken at approximately noon each day (x-axis)

F I G U R E 3
Post-mortem computerized tomographic measurement of pulmonary vascular volumes with representative images (a = Sham, b = PE, c = PE+SU5416, d = PE+SU5416+CINA; no image available for PE+SU+RIO). CINA, cinaciguat; PE, pulmonary embolism with microspheres alone; RIO, riociguat. The asterisk indicates significance at p < 0.05 from Tukey's post hoc following one-way ANOVA, versus all other groups

| SU5416 inhibits endothelial tube formation on Geltrex; rescue by CINA
A preliminary dose-response experiment indicated that 25 μM SU5416 was required to inhibit PMVEC or PAEC tube formation of on Geltrex (data not shown). Some disruption of tube structures was observed at SU5416 concentrations of 0.25 and 2.5 μM, but these were not statistically significant when images were tested by the Angiogenesis Analyzer (AA) plug-in for Image J (data not shown). Phasecontrast images of endothelial tube networks formed on Geltrex are shown in Figure 7a,c. The disruptive effect of SU5416 on tube networks by both PMVECs and PAECs is evident in the DMSO control versus SU5416 images qualitatively and quantitatively in Figure 7a-d. Co-treatment with 10 μM CINA, but not 10 μM RIO, improved tube networks in both cells. Image J quantitates 20 metrics of tube formation with varying amounts of sensitivity (ability to distinguish small differences in tube network complexity). SU5416 disrupted 10 and 19 of these metrics for PMVECs and PAECs, respectively (not shown). When cells were co-treated with SU5416 plus CINA, 10 metrics each were improved for PMVECs and PAECs, while RIO improved only one metric for PMVECs and none for PAECs (not shown). Quantitation of the SU5416, CINA and RIO treatments by Image J AA is shown in Figure 7b,d for select AA metrics for which SU5416+CINA was significantly improved versus SU5416 alone. Increasing the RIO concentration in this assay from 10 uM to 100 uM did not result in improved tube formation (data not shown).

| DISCUSSION
Treatment with the sGC stimulator and activators CINA and RIO both ameliorated the development of the CTEPH phenotype in rats with CPE accompanied by in vivo and in vitro evidence of enhanced angiogenesis. Metrics of right ventricular function were improved by both compounds, including RVSP, exercise tolerance, and right ventricular hypertrophy. These results support the use of sGC modulators to ameliorate the progression from CTED to CTEPH in human patients with PE. The beneficial effects of sGC agonists were accompanied by increased pulmonary angiogenesis in vivo, implying a causal link. This linkage was supported by several lines of evidence. First, aortic and pulmonary artery rings derived from rats treated with CINA or RIO had higher angiogenic potential when cultured in Geltrex than rings from rats with CPE but no CINA or RIO treatment. Second, CINA, but not RIO, improved endothelial tube formation on Geltrex basement membrane formulation in pulmonary endothelial cells treated with SU5416. Both of these results suggest that sGC agonists "rescue" the angiogenic potential of pulmonary endothelial cells from the VEGF/ VEGF-R2-signaling inhibition caused by SU5416. It is unclear why RIO did not rescue endothelial tube formation on Matrigel, even at an elevated dose of 100 μM, which was cytotoxic (data not shown). RIO was active in stimulating sGC activity in whole tissues extracts of rat lung with cGMP production as an endpoint (data not shown). This contrasts with the pro-angiogenic effect RIO had on rats in vivo and the increased angiogenesis we observed in ex vivo aortic and PA rings taken from RIO-treated rats ( Figure 6).
We propose that angiogenesis occurs from the normal healing response to persistent PE, as is the case with the present model, which occurs in two phases: acute inflammation followed by angiogenesis (Ridiandries et al., 2016). Acute PE in humans and animals produces a robust inflammatory response within hours, characterized by increase in transcription and expression of chemokines in the lungs and increased circulating concentrations of interleukins, followed by cellular infiltration in the lung and heart (Nakos et al., 1998;Stewart et al., 2015;Zagorski et al., 2003Zagorski et al., , 2007. Our data in rats show that the lung switches from an inflammatory phenotype to an angiogenic phenotype starting a few days after PE, and that disruption of VEGF signaling with the VEGF-R2 inhibitor F I G U R E 6 Phase-contrast images of aortic (Ao; top 3 lines) and pulmonary artery (PA; bottom 3 lines) rings. Aortas and pulmonary arteries were dissected from rats 4 weeks after treatments to produce CPE. Rings were embedded in Geltrex and over-laid with basal M1266 medium plus glutamine, antibiotic-antimycotic, and 0.1% FBS but lacking growth factors (EGF, VEGF). After 10 days rings were labeled with MTT to enhance visibility of sprouts and fixed with 2% formaldehyde. Representative images are shown for five rats each from groups of 8-10 CPE rats treated with either cinaciguat (CINA), RIO, or DMSO. Aortas and PAs were obtained from the same rat in each treatment group SU5416, reliably causes severe PH post-PE (Neto-Neves et al., 2017). Angiogenesis refers to the sprouting of new vessels from existing vessels, driven by extension and proliferation of ECs (Stapor et al., 2014). The growth factor VEGF has been called a master regulator of both the growth and maintenance of pulmonary vessels, although VEGF-driven angiogenesis may even require other cytokines and growth factors, notably PDGF (Heldin et al., 1998;Hellberg et al., 2010;Voelkel & Gomez-Arroyo, 2014). In animal models, VEGF is increased in experimental thrombi and contributes to clot resolution (Waltham et al., 2000(Waltham et al., , 2003. SU5416 is well known to cause PH together with ambient hypoxia (Voelkel & Gomez-Arroyo, 2014). The role of disrupted VEGF signaling as a cause F I G U R E 7 Geltrex tube assays of rat endothelial cells treated with SU5416 plus cinaciguat (CINA) or riociguat (RIO). Panel a: Phasecontrast images of representative wells from triplicate assays of pulmonary microvascular endothelial cells (PMVECs) (25k/well) treated with the indicated supplements. Control cells were incubated with M1266 basal medium containing glutamine and antibiotic-antimycotic mix without growth factors or serum. DMSO was added at a concentration of 0.2% in control samples. SU5416, CINA, and RIO dissolved in DMSO were added to M1266 basal medium at the indicated concentrations. Panel b: Angiogenesis Analyzer metrics of PMVECs treated in Panel a. Representative metrics in which SU5416+CINA treatments were significantly different from SU5416 alone are shown (p < 0.05, ANOVA with Tukey's pairwise test). Panels c and d, same as a and b except PAECs (20k/well) of PH post-PE remains largely hypothetical. About 20% of humans have alternative splicing that alters VEGF signaling, but the epidemiological understanding of genetic deficiencies in VEGF signaling axis related to CTEPH is nascent. As an example, alternative splicing of the VEGF decoy receptor VEGF-R1, sometimes called fms-related tyrosine kinase (flt-1), results in a soluble variant (s-Flt1) that can sequester VEGF and decrease its activity (Kendall & Thomas, 1993). In small studies, patients with CTEPH had elevated circulating concentrations of s-Flt1 (Saleby et al., 2017;Tiede et al., 2015). The role of antiangiogenic splice variants in the VEGF-A protein (the VEGF xxx b variants) remain poorly characterized in CTEPH (Suzuki et al., 2016). CTEPH patients have elevated blood concentrations of VEGF, and increased expression of angiostatic receptor CXCR3, especially in distal lung arteries (Saleby et al., 2017;Zabini et al., 2012). Lung tissues from CTEPH patients demonstrate increased VEGF in regions of lung and in thrombus that are dense in new vascular growth, but not in fibrotic areas (Bochenek et al., 2017). Taken together, available data suggest that many patients with CTEPH have either inherited or acquired defects in VEGF signaling, although it is clear neither VEGF's action nor its disruption is the sole mediator of either the normal angiogenic response or progression to PH, respectively.
Guanylate cyclase modulators may overcome inhibition of angiogenesis produced by the combination of PE and VEGF antagonism at the post-receptor level. At the subcellular level, VEGF-R2 is the primary receptor tyrosine kinase (RTK) responsible for angiogenesis, and it activates the cytosolic kinases ERK-1 and -2, which have multiple effects. When phosphorylated via VEGF-R2 signaling, the ERK1/2 isoforms increase endothelial cell growth, proliferation, survival, and phosphorylation of PFKFB3 (Novellasdemunt et al., 2012(Novellasdemunt et al., , 2013, the key regulator of glycolytic flux. This is important because glycolysis supplies cytosolic ATP necessary for endothelial pseudopod formation and tip cell extensions required for new vessel growth (Stapor et al., 2014). ERK1/2 phosphorylation can also be induced by increases in cellular cGMP mediated by the enzyme soluble guanylate cyclase (sGC) (Pyriochou et al., 2006). These data formed the rationale for the hypothesis that sGC modulation could increase angiogenesis in a tandem model of CTEPH.

| CONCLUSIONS
In rats with chronic pulmonary hypertension produced by PE followed by treatment with the RTK inhibitor SU5416, sGC stimulation and activation prevents subsequent PH, coincident with increased pulmonary angiogenesis. These data suggest a role for pharmacological sGC modulation in the acute post-PE period to reduce probability of the chronic PH phenotype.

ACKNOWLEDGMENTS
The authors thank Maria Zaretsky for her technical expertise on this work and also thank other members of the Emergency Medicine Lab for helpful discussions and comments (Karen Zhou MD, Ziqian Zeng, and Tanmaye Nallan Chakravarthula). Lung CT imaging was done by Paul R. Territo, PhD, IU Medicine, and colleagues. Paul Herring, PhD (IU Department of Cellular and Integrative Physiology) graciously allowed the use of his cell culture lab.

CONFLICT OF INTEREST
CINA was provided as an unrestricted in-kind donation from Bayer. The authors have no other financial conflict to report.

AUTHOR CONTRIBUTIONS
John Zagorski co-conceived the work, collected and analyzed the data, and wrote the first draft of the manuscript. Evandro Neto-Neves developed the model system, collected data, and edited the manuscript, Nathan J. Alves provided technical and scientific direction and edited the manuscript. Amanda J. Fisher performed surgeries, collected data, and edited the manuscript and Jeffrey A. Kline co-conceived the work, obtained funding, collected and analyzed the data, and co-wrote the manuscript.


Protective Effects of Polydatin on Reproductive Injury Induced by Ionizing Radiation

The reproductive system is vulnerable to ionizing radiation, which is a hot research topic at present. We tested the effect of polydatin on spermatocytes(GC-1 cells) after X-ray irradiation. The reproductive damage model of C.elegans was established by 60Coγ-ray, and the protective effect of polydatin on reproductive damage caused by ionizing radiation was evaluated. We quantified the ROS levels of GC-1 cells and C.elegans after irradiation with polydatin and evaluated the anti-apoptosis effect of polydatin at proper concentration. Differential genes of C.elegans reproductive damage were screened out from transcriptome sequencing results and comparable GEO datasets. It was proved that 100μM polydatin significantly reduced the apoptosis of GC-1 cells induced by 2 Gy X-ray. In addition, the longevity, reproductive capacity, germ cell apoptosis and spawning and hatching capacity of polydatin were tested. The results showed that 100 μM polydatin content significantly increased the influence of 50 Gy 60Coγ-ray on reproductive capacity of C.elegans. Quantitative analysis of mRNA and protein levels of apoptosis-related genes and reproductive-related genes by qRT-PCR and Western blotcon firmed that polydatin with appropriate dosage had good protective effects on reproductive damage caused by radiation, which laid a foundation for the application research of polydatin in radiation protection.

Introduction
Ionizing radiation technology has been widely used in daily life, but the accompanying risk of radiation damage is also increasing. The reproductive system is a sensitive target organ of ionizing radiation. Excessive radiation will affect reproductive function, resulting in infertility, exposure during pregnancy will also cause embryo deformity, abortion, and other adverse outcomes. 1,2 The epidemiological results of the Chernobyl accident found that long-term low-dose radiation increased the risk of infertility or postpartum anemia among women, 3 cohort study in high background radiation area found long-term low dose exposure to reduce male sperm motility. 4 These radiation-induced reproductive health problems plague scientific research workers, and it is urgent to find appropriate radiation protection agents and explore its biological mechanism.
Because of the reproducibility of sperm and spermatogonia, spermatogonia are usually used by researchers in germ cell selection experiments or directly detecting sperm motility. 5 Among them, GC-1 cells of mouse spermatogonia played an important role in the study of reproductive damage mechanism caused by different types of radiation. 6,7 In addition, due to the particularity of the reproductive system, it is particularly important to choose the appropriate model organism to study reproductive damage. In recent years, researchers have found that Caenorhabditis elegans(C.elegans) is a reliable model organism for studying reproductive damage, aging, and nervous system damage. [8][9][10] In the study of radiation-induced reproductive damage, ionizing radiation can inhibit C.elegans reproductive ability, increase the level of ROS in vivo, and cause apoptosis of germ cells, which is a good biological model to study radiation-induced reproductive damage. [11][12][13] And provides a scientific reference basis for researchers.
Currently, there are several anti-radiation drugs available in clinics with definite efficacy, such as amifostine, glycoprotein granulocyte colony-stimulating factor (G-CSF), and nitroxide. 14 However, these drugs have adverse effects and low safety profiles, and their effects on the reproductive system are elusive. [15][16][17] Therefore, there is an urgent need to identify new and effective agents that can protect against radiation-induced damage to the reproductive system. Polydatin, the main active ingredient of Polygonum cuspidatum, is a natural precursor of resveratrol and has similar pharmacological effects as resveratrol. 18 However, polydatin has a stronger antioxidant activity and higher stability than resveratrol. 19 Studies have shown that polydatin can significantly inhibit the production of excessive oxygen free radicals and reactive oxygen species in vivo. 20 Ma et al. 21 results showed that polydatin inhibited the apoptosis of germ cells and reduced the damage of ionizing radiation to testis by scavenging ROS in C57BL/6 mice. Moreover, polydatin can improve the damage of hematopoietic system caused by neutron radiation by inhibiting cell apoptosis. 22 The above evidence shows that polydatin has a good prospect in radiation protection, and its reliability needs more experimental verification.
Based on the above reasons, this study used X-ray to irradiate GC-1 cells, and 60 Coγ-ray was used to establish the radiation reproductive damage model of C.elegans and tested the influence of polydatin on various indicators of C.elegans reproductive ability after irradiation. The protective effect of polydatin on radiation-induced reproductive injury was verified by detecting apoptosis-related genes and reproduction-related genes. It is hoped that our research will provide scientific basis for exploring the feasibility of polydatin as a potential new radioprotective agent against reproductive injury.

Cell Culture
BALB/c mouse sperm cell-derived cell line (GC-1) and cellspecific medium CM-0600 were purchased from Pro cell Life Technology Co., Ltd. of Wuhan, China. Culture at 37°C and 5% CO 2 . For the purpose of each experiment, the number of cells was determined.

C.elegans Culture and Grouping
The wild-type N2 C.elegans worms and Escherichia coli OP50 (E. coli OP50) were kindly provided by Prof. Huimin Zhang, from the School of Basic Medicine and Life Sciences, Soochow University. The worms were cultured on NGM agar medium containing E.coli OP50 at 20°C. 23 According to the irradiation dose, C.elegans were divided into two groups: 0Gy (control group) and 50Gy group. Twenty C.elegans were observed and recorded in each group.

Synchronization of the Worms
Adult C.elegans worms were collected using M9 buffer and incubated in a bleach solution to synchronize them at the L1 stage as described by Shen et al. 24 After 28 h of culture at 20°C, these worms entered the L4 stage. For the synchronization of the descendant worms, the female worms in the oviposition period were transferred onto new NGM culture dishes for 2 h after oviposition, and then cultured at 20°C for 37 h to enter the L4 phase.

Irradiation Conditions
L4-stage C.elegans worms were irradiated with 50 Gy of radiation at a rate of 25 Gy/min at 20°C using a 60 Co-γ irradiation device from the State Key Laboratory of Radiation Medicine and Protection and School of Radiation Medicine and Protection, Soochow University. When the cells reached the logarithmic phase, GC-1 cells were treated with 1, 2, 3, and 4 Gy biological X-ray machines (Rad source, Inc.). Under sterile conditions, the appropriate dose was screened at the dose rate of 1 Gy/min.

Drug Intervention
GC-1 cells and C.elegans intervention of resveratrol glycoside is to add sterile resveratrol glycoside to the culture medium in the pre-irradiation stage. 25 The drug concentrations were set to 0 μM (control group), 50 μM, 100 μM, and 200 μM, respectively. The control group was replaced by normal saline with the same dose.

Assessment of C.elegans Life Expectancy
Measurement life reference 26. After the intervention of resveratrol glucoside, they were transferred to new plates, each of which was 10. The target C.elegans will be picked in new incubators every day to prevent the influence of newborn larvae, until all C.elegans are observed to die based on no response to contacting C.elegans with the picker. 50 C.elegans were observed in each group.

Determination of the Number of Offspring
The worms were collected and washed with M9 buffer after treatment with polydatin. Each worm was transferred onto a new OMG plate containing OP50 and cultured at 20°C, renewing the plates every 24 h. All eggs and larvae on the plates were counted. The number of unhatched eggs was counted on the next day. After oviposition, the numbers of total and hatched eggs were counted to determine the hatching rate as calculated by the ratio of the number of live eggs to the total number of the eggs laid by each worm. Thirty worms per group were evaluated.

ROS Level Detection
After the cells in each group were treated and cultured to about 80% of the cell density, DCFH-DA staining solution was added according to the instructions of ROS detection kit, and incubated for 30 minutes in an incubator at 37°C. Five 40-fold fields of vision were randomly photographed and recorded under the fluorescence microscope. Fluorescence intensity was quantitatively analyzed by Image J. The experiment was repeated three times.
After irradiation, the warms were washed with M9 buffer and placed in a 1.5-mL Eppendorf tube. They were stained with 10 μM H2DCFDA in 1 mL of M9 buffer for 30 min at 20°C. Subsequently, they were washed 2-3 times with M9 buffer to reduce the background in fluorescence microscopy. Next, the worms were placed onto a glass slide with 2% agarose gel, anesthetized with 30 μL of levamisole, and covered with a cover glass. Images were captured with a fluorescence microscope at an excitation wavelength of 488 nm and a blocking wavelength of 515 nm. Thirty worms per group were examined.

Determination of Cells Apoptosis
GC-1 cells in logarithmic phase were inoculated into 6-well plates at 2 mL per well (2 × 10 6 cells/mL). The AnnexinV-FITC kit was used to detect apoptosis. Single staining of PI and accessories was carried out in V-FITC and incubated for 5 minutes in the dark at room temperature with PI and accessories in V-FITC as controls. Within 1 hour, the fluorescence intensity was detected by flow cytometry, and the experiment was repeated three times to analyze the apoptosis.
The method of Kelly et al. 27 was used to measure the apoptosis of germ cells in C.elegans. Briefly, irradiated worms were washed with M9 buffer, placed in a 1.5-mL Eppendorf tube, and centrifuged at 1500 rpm for 2 min, and the supernatant was discarded. The worms were stained with a 100-μL staining solution containing 25 μg/mL acridine orange and incubated with 5 μL of OP50 for 1 h in the dark. They were then transferred to NGM medium without food for 2 h. Next, the worms were placed onto a glass slide and examined under a fluorescent microscope as indicated above. Thirty worms per group were evaluated.

Transcriptomics Sequencing and Bioinformatics Analysis
We selected C.elegans with radiation-induced reproductive defects and C.elegans in Adult phase control group of the same age after 60 Go-γ irradiation with 100Gy for RNA-seq analysis by Shanghai Europe Easy Co., Ltd. to obtain a transcriptomics sequencing. In addition, the gene expression profile of C.elegans GSE122996 after 200Gy irradiation was extracted from the GEO database, and the differentially expressed genes were analyzed by online GEO2R. The screening criteria of fold change＞2.0 and P-value<.05 were adopted for both transcriptome and GEO datasets. Using David online analysis tool (https://David. ncifcrf.gov/home.jsp) for KEGG analysis and GO analysis of Differentialy Expressed Genes(DEGs) appeared in the two groups of data were conducted.

Screening of Hub Gene
A online database STRING (https://string-db.org/) was selected to analyze protein-protein interaction (PPI) in DEGs, visualize PPI networks and identify hub genes in differential DEGs. The MCC method in CytoHubba, a plug-in Cytoscape, was used to screen hub genes in TOP 40, and the important gene modules were classified according to the MCODE plugin. According to the default parameters, 28,29 the pivotal genes in modules with high scores were screened out. The combination of genes obtained by the two methods laid a foundation for further experimental verification.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
The total RNA in GC-1 cells and C.elegans was extracted by Trizol, and the cDNA was obtained by reverse transcription. After the Real-time PCR reaction system was prepared, the amplification reaction was performed on a PCR instrument. The relative multiple ratio of mRNA expression in the experimental group and the control group was expressed as Folds=2 -44Ct . The experiment was repeated three times, and the average value was calculated. See Supplement Table S1 for primers.

Western Blot
Cells in each group were collected after treatment under different conditions. Subsequently, the cells were lysed on ice with RIPA lysis buffer for 30min. The lysates were centrifuged at 1200r/min for 5 min at 4°C, the supernatant was collected, and protein was quantified by BCA method. Then denatured with loading buffer at 100°C, for 10 min with 10μg per lane for SDS-PAGE. The isolated protein was transferred to PVDF membrane within 120 min at a constant voltage of 300mA. 5% skim milk was used to block non-specific binding at room temperature. After 1.5 h, the membranes were sequentially exposed to primary (4°C, overnight) and secondary (37°C, 1.5 h). ECL solutions were prepared to form protein bands and the chemiluminescent imaging system was operated to capture images. The differences of protein expression were analyzed by image J.

Statistical Analysis
Measurement data in mean ± standard deviation were analyzed on SPSS 20.0 statistical software(IBM). For statistical analysis between two independent samples, independent sample t test(t 0 test used in heterogeneity of variance), one-way ANOVA test for multi-group results and LSD-t test for intergroup significance analysis are used. P < .05 indicated that the difference was statistically significant. Each experiment was repeated at least three times.

Effect of different radiation doses and concentrations of polydatin on proliferation of GC-1 cells
From the results, we can see that when the dose of X-ray reaches 2Gy, the proliferation of GC-1 cells is significantly affected, and it is statistically significant. In later experiments, 2Gy was selected as the irradiation dose of GC-1 cells, as shown in Figure 1A. Different concentrations of polydatin enhanced the proliferation of GC-1 cells. When the concentration needed to reach 100μM, the cell proliferation was statistically significant with the control group. In the later experiments, 100μM polydatin was used as the interference concentration of GC-1 cells, as shown in Figure 1B.

Effects of polydatin on ROS levels in GC-1 cells induced by ionizing radiation.
Under the fluorescence microscope, it was observed that the control group had the lowest ROS level of GC-1 cells. The fluorescence of irradiated cells treated with 100 μ M polydatin was not significantly different from that in the control group, but the fluorescence of cells in the simple irradiation group was stronger, shown in Figure 2A. Quantitative analysis of the fluorescence value of the photos by Image J revealed that there was no statistical difference in fluorescence intensity between the polydatin intervention group and the control group. The fluorescence intensity of pure irradiation group was higher than that of the control group, and the difference was statistically significant. Compared with the pure irradiation group, the fluorescence intensity of polydatin intervention group decreased, and the difference was statistically significant. The results showed that 100μM polydatin could significantly reduce the level of ROS in GC-1 cells induced by X-ray, shown in Figure 2C.

Effects of Polydatin on GC-1 Cells Apoptosis Induced by Ionizing Radiation
Flow cytometry was used to detect GC-1 cells apoptosis in each group. The apoptosis rate of GC-1 cells in the control group was the lowest and that in the irradiation group was the highest. The apoptosis rate of GC-1 cells in the intervention group of 100μM polydatin was between the two groups, shown in Figure 2B. Quantitative analysis of apoptosis rate by flow cytometry showed that the apoptosis rate of polydatin intervention group was significantly different from that of control group (P < .05). The apoptosis rate of irradiated group was higher than that of control group, and the difference was statistically significant (P < .05). There was a statistically significant difference in the apoptosis rate between the resveratrol intervention group and the radiation group (P <.05). The results showed that 100μM polydatin could significantly reduce X-ray-induced GC-1 cells apoptosis, shown in Figure 2D.

Effects of Different Concentrations of Polydatin on the Life Span of C.elegans
The lifespan of C.elegans is an important indicator for toxicity assessment. 30 As shown in Figure 1, polydatin exerted no suppressive effect on the lifespan of C.elegans. In contrast, 100 μM of polydatin significantly prolonged the lifespan compared with the control (P < .05). No significant difference was observed between the other groups and the control. Therefore, our results showed that 100 μM of polydatin could prolong the lifespan of C.elegans without any marked toxic effect, shown in Figure 3A.

Effects of Different Concentrations of Polydatin on the Reproductive Ability of C.elegans
As shown in Figure 3B, polydatin at 50 μM or 100 μM had no effect on the number of eggs laid compared with that in the  control group. However, at the high dose of 200 μM, polydatin significantly decreased the number of eggs laid compared with the control (P < .05). As shown in Figure 3C, the hatching rate was unaffected when the worms were exposed to polydatin even at 200 μM, suggesting that although 200 μM polydatin decreased the oviposition ability, it had no effect on the survival rate of the embryos. Polydatin at 100 μM had no effect on C.elegans reproductive capacity, including oviposition and embryonic survival. Polydatin significantly reduced the 60 Coγ-induced elevation of ROS levels.
Quantitative analysis of the fluorescence intensity using Image J showed that there was no statistically significant difference in the fluorescence intensity between the 60 Co γ-irradiated group treated with 100 μM polydatin and the control group (P > .05). The fluorescence intensity of the irradiated worms was significantly higher than that of the control group (P < .05). However, polydatin treatment significantly decreased the fluorescence intensity of the irradiated worms (P < .05). These results suggested that 100 μM of polydatin significantly reduced 60 Co γ-induced D: Effect of polydatin on the brood size of C.elegans exposed to 60 Co γ radiation. E: The ratio of the egg incubation periods. *indicated that the difference was statistically significant, P<.05. elevation of ROS levels in C.elegans, as shown in Figure  4A, B.

Polydatin Significantly Reduces the Number of Radiation-Induced Apoptosis of Germ Cells in C.elegans
C.elegans germ cell apoptosis following 60 Co γ irradiation was evaluated through laser confocal microscopy. As shown in Figure 2C, green cells indicated by the arrow are apoptotic cells. There was no significant difference in the number of apoptotic germ cells between 100 μM polydatin-treated and control groups(P > .05) The apoptosis of germ cells was significantly induced by 60 Co γ irradiation compared with the control (P < .05). However, polydatin treatment reduced the number of radiation-induced apoptotic cells (P < .05). These results suggested that 100 μM of polydatin significantly reduced the 60 Co γ-induced germ cell apoptosis in C.elegans. as shown in Figure 4C, D.

Bioinformatics Analysis Results
The results of transcriptomics sequencing and the GSE122996 differential gene by combining 60 Co γ-ray induced reproductive damage in C.elegans are presented in Figure 5A, B. A total of 258 common differential genes were obtained, as shown in Figure 5C. To analyze the biological action and potential function of DEGs, GO analysis including biological process (BP), molecular function (MF), and cellular component (CC) and KEGG signaling pathway enrichment were performed, as shown in Figure 5D. For MF, DEGs was mainly enriched in Regulation of oligodendrocyte Proliferative Growth, tissue morphogenesis, killing of cells of other organism, metal ion transport, and positive regulation of adenosine receptor signaling pathway. For MF, DEGs is mainly enriched in the Thiamine Phosphate Phosphatase Activity, Lysophosphatidic Acid Phosphate Activity, deltacatenin binding, Sodium: Potassium-Exchanging ATPase Activity, and gamma-catenin binding. For CC, DEGs were mainly enriched in perineuronal net, pseudopodium, collagen type VI trimer, endopodestase Clp complex, and Mitochondrial endopodestase Clp complex; Enrichment analysis of KEGG signaling pathway showed that DEGs was mainly enriched in non-homologous end-joining, protein export, homologous reconstitution, oxidative phosphorylation, and metabolism of xenobiotics by cytochrome P450, as shown in Figure 5E. Analysis of all the statistically different apoptosisrelated pathways shows that the pathway of apoptosis process, apoptotic mitochondrial changes and oviposition, and actin filament depolymerizationis were closely related to egl-1. cep-1 that participates in the intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator. These two genes were associated with ionizing radiation-induced apoptosis, egl-1 was homologous to the BH3(Noxa) gene in human and mouse, cep-1 to human TP53 and mouse Trp53.
The pathways involved by these two genes are shown in Supplement Table S2.

Screening of Hub Gene for Reproductive Damage Induced by Ionizing Radiation
The first 40 hub genes were obtained by using cytohHubba plug-in cytoscape3.8.2 software, and the functional modules were screened by MCODE plug-in to select the first module with the highest score. A total of 38 genes were detected by these two methods. Screening of these genes shows that 10 genes belong to MSP sperm major protein family, which includes sperm formation related genes gsp-3 and glf-1, sperm specificity family ssh-1, and cell cycle related gene cyc-2.2, as shown in Figure 5F, G. In order to screen out the hub genes that can be used as biomarkers, in this study, we selected 38 genes with high scores in transcriptome and significant multiple changes. We screened ssh-1, a sperm-specific family gene, and gsp-3, a gene associated with spermatogenesis and meiosis. ssq-1 is closely related to the genes of heat shock protein family A, of which heat shock protein HSPA2 (a gene homologous to mouse Hspa2) is an important biomarker for the detection of male infertility. 31 gsp-3 is homologous to human and mouse PPP1CA(Ppp1ca), and serine/threonine protein phosphatase (PPPs) is involved in the regulation of sperm overactivity. Therefore, these two hub genes were selected as biomarkers to detect reproductive damage induced by ionizing radiation. Pathways for msp-10 and gsp-3 are shown in Supplement Table S3.

Effects of Polydatin on Expression of Regulatory Genes Related to Apoptosis and Reproduction by Ionizing Radiation
The effects of Polygonum cuspidatum on apoptosis regulation and reproductive regulation genes of GC-1 cells after 2 Gy X-ray irradiation are as follows. In the mRNA level analysis results of apoptosis genes BH3 and Trp53, these two apoptosis genes were up-regulated in the radiation group, which was statistically different from the control group. Compared with the control group, the two genes in resveratrol intervention group were up-regulated, but down-regulated compared with the irradiation group alone, with statistical significance, as shown in Fig. A, B. The mRNA level analysis of reproductive related genes Ppp1ca and Tnp2 showed that the expression of Ppp1ca was down-regulated and that of Tnp2 was upregulated in the irradiation group alone and polydatin intervention group could obviously reverse this phenomenon, and there was no significant difference compared with the control group. The results showed that 100μM polydatin could reduce the damage of X-ray to GC-1 cells, as shown in Figure 6C, D.
As shown in Figure 6E and F, in the influence of polydatin on apoptosis regulation and reproductive related regulation genes of C.elegans induced by 60 Co γ-ray, the mRNA expressions of apoptosis regulation genes egl-1 and cep-1 in the irradiation group were significantly higher than that in the control group. The mRNA levels of reproduction-related genes gsp-3 and msp-10 were analyzed and the results were consistent with those of X-ray irradiated GC-1 cells. Resveratrol intervention group could significantly down- regulate the mRNA levels of the reproductive related genes gsp-3 and msp-10 induced by ionizing radiation. These differences are statistically significant ( Figure 6G, H). The results suggested that 100 μM polydatin could significantly improve the 60 Co γ -ray induced apoptosis regulation of C.elegans and the expression of reproductive-related regulatory genes and reduce the apoptosis of C.elegans germ cells.

Effect of Polydatin on Apoptosis of GC-1 Induced by Ionizing Radiation and Expression of Reproduction-Related Proteins
In the detection of protein expression level of Ppp1ca, a reproductive-related gene after polydatin interference, the protein level showed an upward trend compared with the control group and a downward trend compared with the X-ray irradiation group, with statistical differences. All other results are consistent with the effect of polydatin on the apoptosis and reproductive regulation genes of GC-1 after 2 Gy X-ray treatment. It was verified from the protein level that 100μm polydatin could reduce the damage of GC-1 induced by X-ray as shown in Figure 7C, D.

Discussion
The damage of ionizing radiation to the reproductive system has always been a matter of great concern to people, but the existing radiation protective agents also have some defects. In recent years, researchers have been looking for new antiradiation drugs, hoping to make up for the shortage of the current anti-radiation drugs. Polydatin is one of the first innovative antioxidant drugs submitted for clinical research in the United States and China, and it has great application potential in radiation-resistant injury and the treatment of such injury. 32 Study have shown that polydatin not only has the effect of lowering blood lipid, 33 lowering blood sugar, 34 and anti-inflammation, 35 but also can effectively prevent oxidative stress, reduce ROS level, increase SOD level, and prevent cell apoptosis caused by oxidative stress. 36,37 Cao et al. 38 found that polydatin can inhibit epithelial-mesenchymal transition through TGF-β1/Smad3 signaling pathway and reduce radiation-induced pulmonary fibrosis. Li et al. 39 Study have shown that polydatin can increase the microvessel density of the small intestine after ionizing radiation, reduce the apoptosis of intestinal microvascular endothelial cells, and promote the growth and cell repair of intestinal crypts. In addition, polydatin was also effective in protecting the reproductive system from radiation-induced damage, such as infertility and sperm dysfunction, mediated by down-regulation of malondialdehyde and 8-hydroxydeoxyguanosine. 21 At the same time, polydatin can also reduce the damage to the testis of mice caused by ionizing radiation by inhibiting the ratio of apoptosis-related protein Bcl-2/Bax. 40 All the above studies have shown that polydatin has a significant radiation protection effect, but for reproductive damage induced by ionizing radiation, more experimental studies are still needed to demonstrate its effective dose and action mechanism.
GC-1 cells used in this study are currently an important cell for reproductive research and have a high sensitivity to ionizing radiation. 41 And is useful in the treatment of mouse germ cell apoptosis caused by heavy ion radiation induced DNA damage. 42 And hMSH5 C85T polymorphisms play an important role in differential studies of sperm production abnormalities induced by ionizing radiation. 43 The germ cells of C.elegans are also very sensitive to ionizing radiation and are widely used to study radiation-induced germ cell apoptosis. 44 We have previously demonstrated that 50 Gy of 60 Coγradiation induces significant damage to the reproductive system of C.elegans, which is more pronounced at doses up to 100 Gy, 45 so in order to better study the genetic changes caused by ionizing radiation-inducedeproductive damage, we selected C.elegans that induced reproductive damage at the dose level of 100 Gy for transcriptomics sequencing and obtained the genes related to ionizing radiation-induced reproductive damage. At the same time, in order to verify the expression of our transcriptome sequencing results in C.elegans exposed to ionizing radiation without reproductive damage, we selected C.elegans with 200 Gy radiation for comparative analysis in GEO database, and found out the genes with common expression. Bioinformatics analysis was used to screen reproductive development-related pathways and apoptosis-related pathways, and hub gene is used to screen human and mouse genes for verification. It improves the scientificity and feasibility of using these genes to verify the protective effect of polydatin on the GC-1 cells damage and reproductive damage of C.elegans induced by ionizing radiation.
Polydatin can improve mouse follicular stability, telomerase activity and offspring survival rate. 46 In this study, we found that 100μM polydatin treatment greatly increased the hatch ability and embryo survival rate of C.elegans and improved the effect of 60 Coγ-ray on their reproductive ability. These results preliminarily proved that polydatin had a protective effect on radiation-induced reproductive injury. Simultaneously, 100μM polydatin increased the lifespan of C.elegans. This result is consistent with the previous discovery that polydatin can prolong the life of mice and C.elegans. 47,48 Ionizing radiation can affect the function of cells by increasing the level of ROS, and increasing the level of ROS will accelerate the apoptosis of cells, thus inducing diseases and even cancer. [49][50][51] Polydatin can eliminate ROS in vivo, reduce insulin-like growth factor À1, increase insulin sensitivity and AMPK activity, increase the number of mitochondria, and improve motor function, with good effects. 52 In addition, many studies have shown that ionizing radiation can accelerate the production of ROS in germ cells, leading to the death and functional damage of germ cells. 53,54 Our results demonstrated that polydatin significantly reduced the ROS levels in GC-1 cells and C.elegans induced by ionizing radiation, and decreased the rate of germ cell apoptosis. These results once again proved the protective effect of polydatin on radiationinduced reproductive damage.
It is well known that TP53 (p53) is a major gene regulating apoptosis, and BH3 is a priming factor for the apoptotic process. It has been found that in the irreversible DNA damage reaction, p53 promotes the BID nucleus output of BH 3-only to induce apoptosis, 55 and plays a key role in the selective attenuation of DNA damage-induced neuron apoptosis by mithramycin 56 and regulation of gingival protease-induced osteoblast apoptosis. 57 In addition, p53-BH3 pathway provides a new direction to promote apoptosis of ovarian cancer cells in gene therapy. 58 Horvitz 59 found that ionizing radiation induced apoptosis of C.elegans germ cells through the cep-1/ egl-1 pathway, and these two genes were similar to p53 mediated BH3 domain protein found in mammals. 60 In addition, the bioinformatics analysis of the transcriptome sequencing results in this study also revealed that these two genes were involved in many ways of regulating apoptosis. The results of this experiment showed that polydatin could reduce the apoptosis of germ cells induced by ionizing radiation by regulating the expressions of Trp53 and BH3 in GC-1 cells and the expressions of cep-1 and egl-1 in C.elegans.
Msp-10, the major sperm protein gene of C.elegans, is a reproduction-related gene unique to this model organism, located on chromosome IV. It is found that sperm cells of nematodes lose actin and tubulin after meiosis, and then use the regulatory assembly and disassembly of the major sperm protein (MSP) to drive sperm movement, which plays an important role in inducing oocyte maturation. 61 And msp-10 may serve as a biomarker for detecting exposure to low dose ionizing radiation during nuclear waste disposal. 62 TNP2 is a unique gene of mammalian sperm development, located on chromosome 16. In the Tnp2-deficient mice, there were abnormalities in the sperm head, decreased motility, and affected the ability of the sperm to penetrate the zona pellucida of the egg cell in the acrosome reaction. 63 In addition, the mRNA level of TNP 2 in male smokers decreased, the motility of sperm decreased, and the morphology of sperm increased abnormally. 64 The gene gsp-3 related to spermatogenesis and meiosis is homologous to human PPP1CA (mice Ppp1ca), which is the catalytic subunit of phosphoprotein phosphatase 1(PP1) and has been proved to be a key regulator of sperm development and function. 65 Moreover, when the PPP1CC subunit is interfered with, the PPP1CA subunit can compensate for this effect, serving as an important pathway involved in sperm formation and a hot topic in the research on male contraceptive programs. 66 There are also PPP1CA, breast cancer, 67 lung cancer, 68 rectal cancer, 69 and others. At the same time, Wu et al. 70 proved that gsp-3 was necessary for the growth and sperm motility of C.elegans. In addition, gsp-3 is closely related to the meiosis of sperm, and can be combined with MEMI and participate in the fertilization process. 71 Our research shows that polydatin can reduce the damage of GC-1 cells by ionizing radiation by regulating the expressions of Tnp2 and Ppp1ca and reduce the damage of germ cells by ionizing radiation by regulating the expressions of gsp-3 and msp-10, thus playing an important role in sperm formation and germ cell development.
In conclusion, the radiation protection effects of 100μM polydatin on reproductive damage of GC-1 cells after irradiation with 2Gy X-ray and 50Gy 60 Co γ-ray was confirmed in our research at the level of cell and model organism C.elegans. Reduce the apoptosis of germ cell by relieving the increase of ROS caused by ionizing radiation; The lifespan, reproductive ability and egg laying rate of C.elegans are detected, and the radiation protection effect of polydatin with the concentration is demonstrated. In addition, the expression levels of apoptosis-regulating genes BH3 (egl-1) and Trp53 (cep-1), the reproduction-related gene Ppp1ca (gsp-3), the mammalian spermatogenesis-specific gene Tnp2 and the C.elegans sperm major protein gene msp-10 were detected. The above results indicated that 100μM polydatin had a good protective effect on radiation-induced reproductive damage, which provided a scientific basis for future research. Therefore, research of polydatin as a new drug for resisting radiation-induced reproductive damage is still going on.

Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundation of China

Supplemental Material
Supplemental material for this article is available online.


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Polyploidization of Hepatocytes: Insights into the Pathogenesis of Liver Diseases

Polyploidization is a process by which cells are induced to possess more than two sets of chromosomes. Although polyploidization is not frequent in mammals, it is closely associated with development and differentiation of specific tissues and organs. The liver is one of the mammalian organs that displays ploidy dynamics in physiological homeostasis during its development. The ratio of polyploid hepatocytes increases significantly in response to hepatic injury from aging, viral infection, iron overload, surgical resection, or metabolic overload, such as that from non-alcoholic fatty liver diseases (NAFLDs). One of the unique features of NAFLD is the marked heterogeneity of hepatocyte nuclear size, which is strongly associated with an adverse liver-related outcome, such as hepatocellular carcinoma, liver transplantation, and liver-related death. Thus, hepatic polyploidization has been suggested as a potential driver in the progression of NAFLDs that are involved in the control of the multiple pathogenicity of the diseases. However, the importance of polyploidy in diverse pathophysiological contexts remains elusive. Recently, several studies reported successful improvement of symptoms of NAFLDs by reducing pathological polyploidy or by controlling cell cycle progression in animal models, suggesting that better understanding the mechanisms of pathological hepatic polyploidy may provide insights into the treatment of hepatic disorders.

INTRODUCTION
Polyploidization is a process by which cells are induced to possess more than two complete sets of chromosomes or become polyploid. Whole-genome polyploidy is common among plants, insects, fish, and amphibia; however, in mammals, polyploidy is restricted in certain cell types including hepatocytes and mammary alveolar cells (Wertheim et al., 2013). Polyploidy of these cells results from both nuclear polyploidy, defined as an increase in the amount of DNA per nucleus, and cellular polyploidy, defined as an increase in the number of nuclei per cell .
Polyploidy was first recognized almost a century ago, and it has been generally considered as a consequence of adaptation during evolution (Comai, 2005). In mammals, the formation of polyploid cells is only observed in certain tissues, which are those that undergo development and differentiation, such as heart, placenta, bone marrow, pancreas, and liver (Pandit et al., 2013). Especially, polyploidy in the heart, kidney, and liver was found during regeneration and repair processes (Cao et al., 2017;Lazzeri et al., 2018;Donne et al., 2020). A high frequency of polyploidy has been detected as a consequence of aging in vascular smooth muscle cells, which is accompanied by symptoms of vascular smooth muscle hypertrophy and hypertension (Hixon and Gualberto, 2003). By contrast, unscheduled polyploidization has been observed in pathological conditions. In murine models of non-alcoholic fatty liver disease (NAFLD), the parenchyma of fatty livers displayed alterations of the polyploidization process. Biopsies from patients with non-alcoholic steatohepatitis (NASH) revealed the presence of alterations in hepatocyte ploidy compared with tissue from control individuals (Gentric et al., 2015). Polyploid and aneuploid cells are frequently seen in various solid tumors, probably because proliferating polyploid cells generate aneuploid daughter cells, leading to genomic instability, which is a risk factor for carcinogenesis (Davoli and Lange, 2011).
In this review, we summarize the current understanding of cellular and molecular mechanisms of polyploidization, and the importance of polyploidy in the normal physiology of the liver. In addition, we review the potential roles of polyploidy in pathological conditions of the liver including NAFLDs, and their implications for treatment of these hepatic disorders.

CELLULAR AND MOLECULAR MECHANISMS OF POLYPLOIDIZATION
Most polyploidization arises from abnormal cell-cycle processes, except for cell fusion, which is a cell-cycle-independent process. To date, three different mechanisms have been proposed for cell-cycle-mediated polyploidization: endoreplication, mitotic slippage, and cytokinesis failure (Gentric and Desdouets, 2014) (Fig. 1).
First, endoreplication, also referred to as endoreduplication or endocycling, is a cell cycle in which DNA is replicated through alternating gap and S phases, resulting in the formation of a mononucleated polyploid cells (Fox and Duronio, 2013). Trophoblast giant cells are the best-known example of endoreplication in mammals. The cells increase their DNA content to attain a large cell size to form a barrier between the maternal and embryonic tissues (Sher et al., 2013). Accumulation of the cyclin kinase inhibitors such as p57/Kip2 was proposed as a part of the mechanism of endoreplicationmediated cell growth and differentiation of trophoblast stem cells; however, the mechanism has not yet been fully elucidated (Ullah et al., 2008). DNA damage is also responsible for the endoreplication in specific species, cell types, and conditions. For example, UV irradiation causes double-strand breakage-triggered G2/M arrest, which is associated with en-doreplication (Radziejwoski et al., 2011). Blockade of mitotic entry after G1/S progression was suggested as an important step leading to endoreplication. Degradation of key regulatory proteins controlling the S phase, such as CDK2, or inhibition of M phase progression factor CDK1, were suggested to cause blockade of mitotic entry (Fox and Duronio, 2013). Second, polyploid cells can be formed after a prolonged arrest in metaphase due to the activation of the spindle assembly checkpoint, which is called mitotic slippage. Subsequently, the perturbed metaphase-anaphase transition causes cell death following mitotic arrest, or mitotic slippage without undergoing anaphase and telophase, which progresses to form mononucleated polyploid cells (Gentric and Desdouets, 2014). Lastly, cytokinesis failure generates binucleated polyploid cells. Cytokinesis is the final step in cell division, leading to the physical separation of a mother cell into two daughter cells. Some cells are programmed to process cytokinesis failure during normal development. For example, hepatocytes exhibit destined cytokinesis failure, which generates multinucleated polyploid cells .

POLYPLOIDY ASSOCIATED WITH NORMAL PHYSIOLOGY IN THE LIVER
The liver is well-known to display ploidy dynamics in physiological homeostasis during its development (Gentric and Desdouets, 2014;Wang et al., 2017). All hepatocytes are diploid at birth, but the cells undergo polyploidization, leading to the gradual heterogeneity of hepatocyte ploidy thereafter. Postnatal binucleation seems to occur during suckling-toweaning transition (about day 21), which is probably a result of insulin-mediated failure of cytokinesis (Celton-Morizur et Biomol Ther 30(5), 391-398 (2022) Fig. 1. Three alternative cell-cycle modes associated with polyploidy. Most polyploidization occurs during abnormal cell-cycle processes. To date, three different mechanisms have been proposed for cell-cycle-mediated polyploidization. Endoreplication is a cell cycle in which DNA is replicated in two successive S phases without continuing to the M phase, resulting in the formation of a mononucleated polyploid cell. Mitotic slippage is linked to the perturbed metaphase-anaphase transition. Cytokinesis is the final step in mitosis which divides the cytoplasm of mother cell into two daughter cells.
The biological importance of polyploidy in hepatocytes has not been understood clearly. The physiological polyploidy of hepatocytes does not show a clear link to functional abnormality of the liver in that prevention of polyploidization by inactivating atypical E2Fs, important regulators of the mitotic and endoreplication cell cycle (vide infra) had no impact on differentiation, zonation, metabolism, or regeneration in the liver (Lammens et al., 2009;Pandit et al., 2012). Meanwhile, some research groups have addressed roles of polyploidy in the liver. Vinogradov (2007, 2010) showed that gene expression patterns associated with fatty acid metabolism and aerobic respiration were altered in polyploid-associated hepatocytes and suggested a functional role of polyploidy in hepatic adaptation to energy storage, cell survival, and tissue regeneration under stressful conditions. Recently, Richter et al. (2021) analyzed the functional characteristics of 2n and 4n hepatocytes based on a single-nucleus RNA-seq2 method.
Comparing 2n and 4n hepatocytes in the Gene Ontology database and informatics resource showed that 4n hepatocytes were more enriched in pathways involved in lipid, cholesterol, and xenobiotic metabolism, suggesting a potential role of polyploidy in different metabolic potential and position within the hepatic lobule of these cells.

POLYPLOIDY ASSOCIATED WITH PATHOPHYSIOLOGY IN THE LIVER
Although hepatocytes in adult liver rarely enter the cell cycle under normal conditions, hepatocytes possess the potential for proliferation in response to hepatic injury from aging, viral infection, iron overload, surgical resection, and metabolic overload, such as that from NAFLDs. Under these circumstances, compensatory proliferation actively takes place, resulting in marked heterogeneity in hepatic ploidy profile (Fig. 2).
During aging in humans, the rate of polyploid hepatocytes is significantly enhanced. The rate of accumulation of binucleate and polyploid cells is very slow until the age of 50 years, but after that, hepatocyte polyploidization is excessively activated, and cells with polyploid nuclei reach 27% by the age of 86-92 years (Kudryavtsev et al., 1993). Activation of p53-p21 and p16 ink4a -pRB pathways in hepatocyte senescence was suggested as a mechanism of polyploidization during aging in a mouse model (Wang et al., 2014). Recently, polyploid hepatocytes have been demonstrated as the source of cel-

Fig. 2.
Pathological polyploidization in the liver. Although hepatocytes in adult liver rarely enter cell cycle under normal conditions, hepatocytes possess potential for proliferation in response to hepatic injury from aging, viral infection, iron overload, surgical resection, and metabolic overload such as NAFLDs. Under these circumstances, compensatory proliferation actively takes place, resulting in marked heterogeneity in hepatic ploidy profile. Endoreplication, the specialized cell cycle in which mitosis is skipped, is one of the mechanisms producing nuclear ploidy in injured hepatocytes. lular turnover and been suggested as an important contributor to liver maintenance during aging (Matsumoto et al., 2021). However, the mechanisms linking polyploidy and aginginduced hepatic dysfunction are not clearly understood and warrant further investigation.
Chronic liver diseases caused by infection with hepatitis B virus (HBV) or hepatitis C virus (HCV) contributes to hepatic adaptation of polyploidy, probably due to cell-cycle defects. HBV X protein (HBx), a crucial viral factor for HBV replication, is assumed to participate in the development of hepatocellular carcinoma (HCC) (Kew, 2011). HBx led to aberrant polyploidization favoring DNA damage propagation and hepatocyte transformation through DNA-damage-mediated Polo-like kinase 1 (PLK1) activation in HBx transgenic mice (Ahodantin et al., 2019). Furthermore, early transition of G1/S phase and delay of G2/M progression observed in the HBx transgenic hepatocytes eventually led to an increase in the number of mononucleated polyploid cells and decrease in that of binucleated cells, which are representative phenomena of endoreplication (Ahodantin et al., 2019). In the case of HCV, infection with the virus or overexpression of its viral core protein induced defects in the mitotic checkpoint and aberrant chromatin segregation in hepatocytes, which showed frequent hepatic polyploidization accompanied by reduced Rb transcription and enhanced expression of E2F1 and MAD2 (Machida et al., 2009).
The liver is a major iron storage organ; however, it is also susceptible to injury from iron overload (Bonkovsky, 1991). Especially, iron overload is considered as a potential risk factor for hepatocarcinogenesis (Hino et al., 2021). It has been shown that the process of polyploidization during carcinogenesis was greatly accelerated in the liver of mice having received iron dextran (Madra et al., 1995). Iron overload induced cyclin D1, a protein involved in the G1 phase of the cell cycle, in a mouse model with carbonyl-iron supplementation or iron-dextran injection, leading to both hepatomegaly and hepatocyte polyploidization (Troadec et al., 2006). Wilson disease, a genetic liver disease accompanied by hepatic copper overload, has also been associated with increased hepatic polyploidy (Yamada et al., 1998;Muramatsu et al., 2000).
The most widely used model to study liver regeneration and hepatic polyploidy in rodents is partial hepatectomy (PHx) using a surgical resection procedure (Michalopoulos and DeFrances, 1997;Martins et al., 2008). A 70% PHx induced hypertrophy followed by hyperplasia, which caused a decrease in the number of diploid cells and binucleated polyploid cells, but an increase in the number of mononucleated polyploid cells. Mechanisms for pathological and regenerative polyploidization are quite distinct. According to the amount of resected liver, different patterns of cellular proliferation were observed during recovery of liver mass. Upon 70% PHx, all hepatocytes entered the cell cycle and rapidly continued through the S phase, but no hepatocytes entered mitosis, resulting in an increase in the number of polyploid hepatocytes. With a resection of >70% of the liver, severe liver injury occurred, and the regenerative capacity of preexisting hepatocytes was significantly impaired (Miyaoka et al., 2012). In this case, liver progenitor cells were activated and progenitor cell-mediated regeneration was the major process in the recovery of liver mass (So et al., 2020). However, after 30% PHx, remaining hepatocytes did not enter into the S phase; instead, hypertrophy of hepatocytes contributed to recovery of the original mass (Miyaoka et al., 2012).

HEPATIC POLYPLOIDIZATION IN NAFLDS
NAFLDs refer to a broad pathological spectrum of diseases ranging from simple steatosis to NASH, which can lead to cirrhosis and eventually to HCC (Hardy et al., 2016). The distinct heterogeneity of hepatocyte nuclear size is one of the unique features of NAFLD (Nakajima et al., 2010;Aravinthan et al., 2013). The degree of nuclear enlargement in patients with NAFLD is greater than that in normal individuals, and a larger hepatocyte nuclear area is strongly associated with an adverse liver-related outcome, including HCC, liver transplantation, and liver-related death (Nakajima et al., 2010;Aravinthan et al., 2013). The enlargement of nuclear size in hepatocytes is closely associated with polyploidization (Watanabe and Tanaka, 1982). In patients with NAFLDs, enrichment of mononucleated polyploid cells was observed throughout the liver parenchyma (Gentric et al., 2015).
In mouse models of NAFLD such as a high-fat diet-induced, methionine-choline-deficient diet-induced, and ob/ob mice, the proportion of mononucleated polyploid hepatocytes was increased, probably due to inefficient cell-cycle progression through the S/G2 phases. Oxidative stress promoted the appearance of highly polyploid cells, and antioxidant-treated NAFLD hepatocytes resumed normal cell division and returned the liver to a physiological state of polyploidy (Gentric et al., 2015). In mice, the liver-specific deletion of SSU72, a protein phosphatase involved in sister chromatin separation, led to polyploidization through endoreplication in chronic liver diseases such as steatohepatitis and fibrosis (Kim et al., 2016). A specific genetic loss of Cdk1 in the liver yielded increased endoreplication in hepatocytes, which was accompanied by a hepatic phenotype of steatosis and fibrosis in mice (Diril et al., 2012;Dewhurst et al., 2020;Ow et al., 2020). Genetic deletion of E2f7 and/or E2f8 in hepatocytes led to decreased nuclear ploidy and increased G2/M transition, suggesting these genes as central transcription regulators that facilitate DNA endoreplication (Chen et al., 2012). Suppression of E2f8 expression in the liver of zebrafish ameliorated diet-induced obesity and this observation may support the link between polyploidy and dysregulation of fat metabolism, although its relevance to human NAFLD was not well addressed (Shimada et al., 2015). Recently, a nuclear receptor RORα was demonstrated as a negative regulator of the E2f7 and E2f8 transcription. Deletion of hepatic RORα aggravated symptoms of NAFLD and enhanced hepatic polyploidy (Kim et al., 2022). These molecular factors that regulate hepatic polyploidy in diverse models are summarized in Table 1.
Polyploidization is closely linked to multiple pathogenic factors for NAFLDs, such as abnormal lipid metabolism and mitochondrial dysfunction. In murine hepatocytes, polyploid nuclei were negatively correlated with the expression of metabolic genes such as Apoa5, Fabp1, Cyp4f15, and Pck1 (Kreutz et al., 2017). A modular biology approach and genome-scale cross-species comparison revealed that gene expression patterns of diploid cells and polyploid cells were different in that genes associated with lipid metabolism were downregulated in polyploid hepatocytes compared with diploid cells (Anatskaya and Vinogradov, 2010). It is noteworthy that expression of genes encoded in the mitochondrial genome has been negatively correlated with nuclear size to a high degree (Miettinen et al., 2014). Hepatocyte senescence resulting from irreversible cell-cycle arrest correlated closely with fibrosis stage and clinical outcome in patients with NAFLDs. The nuclear area of hepatocytes increased in patients with progressed fibrosis but was unchanged in patients with improved fibrosis (Aravinthan et al., 2013). Cellular senescence was also reported to act as a driver of age-dependent hepatic steatosis and nuclear enlargement was detected in the hepatocytes in senescence (Ogrodnik et al., 2017). Together, these observations suggest that hepatic polyploidization may drive progression of NAFLDs through control of multiple pathogenic factors for the diseases.

POLYPLOIDY AND HEPATOCELLULAR CARCINOMA
The contribution of polyploidization to development of HCC has been controversial to date. It was proposed that polyploidy provides a genetic buffer from mutation of tumor suppressor genes because polyploid hepatocytes contain extra chromosomes that probably contain an intact allele (Wang et al., 2021). Consistently, it was reported that mouse hepatocytes were more proliferative when in diploidy than in polyploidy (Wilkinson et al., 2019). The tumor-suppressive effect of polyploidy was demonstrated using genetically modified animal models. Diethylnitrosamine treatment of mice with deleted Anln, a gene encoding cytoskeletal scaffolding protein that regulates cytokinesis and might promote tumorigenesis (polyploid model) and E2f7/E2f8 knockout mice (diploid model), showed that the polyploid mouse model was more resistant to the development of HCC after treatment with the hepatocarcinogen (Zhang et al., 2018;Wilkinson et al., 2019). Meanwhile, other investigators have presented evidence for an alternative concept, that is, the tumor-promoting function of polyploidy. Bou-Nader et al. (2020) showed that the percentage of mononucleated polyploid hepatocytes in patients was positively correlated with incidence of HCC and poor prognosis. In a murine model, treatment with diethylnitrosamine increased the generation of polyploidy hepatocytes that was accompanied by upregulation of Aurora kinase B (AURKB) (Lin et al., 2021). Further studies are required to address the circumstances and environment that determine the pro-or antitumor effects of polyploidy.

THERAPEUTIC IMPLICATIONS OF TARGETING PATHOLOGICAL POLYPLOIDY FOR HEPATIC DISORDERS
Due to various infections and metabolic abnormalities, there is a high frequency of developing diseases in the liver. Because of the growing epidemic of risk factors such as overnutrition, the incidence of NAFLD has risen sharply in the past three decades worldwide (Benedict and Zhang, 2017). Recently, the demand for drugs to treat NAFLD and the market size for them have been increasing; however, as yet there is no FDA-approved drug to treat NAFLD. The pathogenesis of NAFLDs is diverse and intertwined with lipotoxicity, inflammation, and fibrosis, in which various types of cells interact each other simultaneously and systematically (Kim and Lee, 2018;Peng et al., 2020;Loomba et al., 2021). However, the molecular mechanisms underlying the progression of NAFLDs remain ambiguous and heterogeneous, meaning that the identification of therapeutic targets has been challenging. Recently, many drug candidates targeting pathogenic drivers including lipogenesis, inflammatory response, mitochondrial stress, oxidative stress, and fibrogenesis have been developed (Loomba et al., 2021). Unfortunately, however, they have not achieved their ultimate goals due to insufficient efficacy or unexpected side effects (Neuschwander-Tetri et al., 2015;Ratziu et al., 2016;Mantovani and Dalbeni, 2021). Therefore, identification of new therapeutic targets is warranted to overcome the limits of classical strategies. Although whether polyploidy is a driver of NAFLDs or a consequence of progression of the diseases is controversial, the mechanism of hepatic polyploidy may provide new insights into the development of therapeutic strategies for the treatment of the diseases. Several recent studies in animal models found successful improvement of symptoms of NAFLDs by reducing pathological polyploidy or by controlling cell-cycle progression. Administration of JC1-40, an RORα activator that induced expression of E2F7/8, significantly reduced hepatic nuclear polyploidization and liver injury in a high-fat diet-induced model of NAFLD in mice (Kim et al., 2012(Kim et al., , 2022. Hepatic steatosis was completely abrogated in E2f1-deficient mice with diet-induced NAFLD symptoms (Denechaud et al., 2016). In addition, genetic depletion of gankyrin, an oncogenic regulator of CDK4 and RB, significantly ameliorated liver fibrosis by inhibiting steatosis and blockade of hepatic proliferation in mice (Dawson et al., 2006;Cast et al., 2019). Moreover, administration of PD-0332991, a small compound that inhibits CDK4, prevented NAFLD development and reversed hepatic steatosis (Jin et al., 2016). Polyploidy of hepatocytes is associated with initiation of tumor formation (Lin et al., 2021;Matsumoto et al., 2021). Pharmacological inhibition of AURKB using AZD1152 reduced nuclear size and tumor foci (Lin et al., 2021). Clearly, further investigations are necessary to design novel strategies against pathological hepatic polyploidization to contribute to the development of therapeutics for diverse hepatic disorders including NAFLDs.

CONCLUSIONS
Polyploidy is closely related to both physiological and pathological contexts in the mammalian liver. Recent studies have now revealed not only the role of hepatic polyploidy in regeneration and development of the liver, but also its impact on liver metabolism and cancer. Although there are as yet unresolved controversies in the pathological role of polyploidy during progression of hepatic diseases such as NAFLDs and HCC, several studies have found beneficial effects of reducing pathological polyploidy or controlling cell-cycle progression to improve symptoms of NAFLDs in animal models. Thus, better understanding the mechanisms of pathological hepatic polyploidy and identification of relevant new targets may provide insights into the treatment of hepatic disorders.