Adventitial Fibrosis and Fibroblast Mechanosensitivity Are Shaped by Sex and Hormonal Status in Pulmonary Arterial Hypertension

Abstract

Highlights

What are the main findings?

• SuHx-induced pulmonary hypertension produced sex- and hormone-dependent adventitial fibrosis, driven by distinct mechanosensitivity profiles in pulmonary artery adventitial fibroblasts.

• In vitro hormone-dependent stiffness activation threshold, persistent transcriptional reprogramming after hormone loss, and chromosome-linked stretch responsiveness.

What is the implication of the main finding?

• Fibroblast mechanosensitivity as a novel target for stage- and sex-specific PAH therapies.

Abstract

Pulmonary arterial hypertension (PAH) is marked by vascular remodeling, yet the role of adventitial fibrosis—and its modulation by sex and hormonal status—remains unclear. We examined stage-specific adventitial remodeling and pulmonary artery adventitial fibroblast (PAAF) mechanosensitivity in male, ovary-intact female, and ovariectomized (OVX) female Sprague–Dawley rats with SuHx-induced PAH. Hemodynamics, pulmonary artery histology, and adventitia-specific transcriptional profiling were integrated with in vitro assays of PAAFs exposed to defined substrate stiffness and stretch. All groups developed comparable increases in mean pulmonary arterial pressure, but vascular resistance shift and adventitial fibrosis diverged by sex: intact females showed attenuated increase in pulmonary vascular resistance and transient collagen accumulation, whereas OVX females mirrored the sustained, male-like progression. Extracellular matrix (ECM) gene activation occurred without smooth muscle actin induction, suggesting noncanonical fibrotic pathways. In vitro, intact female PAAFs required higher substrate stiffness to induce profibrotic gene expression, indicating a hormone-modulated stiffness threshold. OVX PAAFs showed persistent transcriptional reprogramming, while stretch-induced ECM upregulation occurred predominantly in male-derived PAAFs. These findings demonstrate that adventitial fibrosis in PAH is shaped by both hormonal and chromosomal sex, independent of hemodynamic severity, and highlight fibroblast mechanosensitivity as a potential target for stage- and sex-specific interventions.

1. Introduction

Pulmonary arterial hypertension (PAH) is characterized by sustained elevations in mean pulmonary arterial pressure (mPAP) exceeding 20 mmHg, leading to extensive vascular remodeling including smooth muscle and fibroblast proliferation, plexiform lesion formation, and vasoconstriction [1,2,3]. A central driver of this remodeling is the dysregulation of the vascular extracellular matrix (ECM), mediated in large part by pulmonary artery adventitial fibroblasts (PAAFs) [4]. These mechanosensitive cells maintain ECM homeostasis under physiological conditions, but in PAH they are exposed to increased environmental stiffness from ECM remodeling, increased cyclic stretch from high intravascular pressures, and other mechanical cues. In this altered environment, PAAFs adopt pathological phenotypes that promote fibrosis and contribute to significant, multi-fold vascular stiffening observed in both animal models and PAH patients [5,6,7].

Our previous work demonstrated that pathological mechanical stimuli—including stretch and substrate stiffness, which are prominent features of PAH—promote profibrotic activity in male-derived PAAFs [5,8]. Clinically, however, PAH occurs more frequently in women [9,10,11], and pre-menopausal women generally have better prognoses than post-menopausal women and men [9,10,11,12]. These observations point to an important role for sex and ovarian hormones in disease progression; however, sex-specific studies of PAAF pathology and mechanobiology remain scarce, and no current therapies explicitly target these differences.

To address this gap, we combined in vivo hemodynamic assessment, ex vivo vascular histology, transcriptional profiling of the adventitial layer, and in vitro PAAF mechanical stimulation to link fibrotic remodeling at the organ-, tissue-, and cellular-levels. We studied male, ovary-intact female, and ovariectomized (OVX) female Sprague–Dawley rats subjected to the sugen-hypoxia (SuHx) model of PAH. We hypothesized that mechano-induced profibrotic signaling in PAAFs and the resulting pulmonary arterial fibrosis are modulated by sex and ovarian hormones, with intact females exhibiting attenuated responses compared with OVX females and males.

2. Materials and Methods

2.1. SuHx Rat Model of PAH

All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, San Diego. Pulmonary hypertension was induced in 7-week-old ovary-intact female (F), ovariectomized female (OVX), and male (M) Sprague–Dawley rats (Charles River, Hollister, CA, USA), weighing 210 ± 26 g, using the sugen-hypoxia (SuHx) model. Ovariectomy was performed by Charles River Laboratories under their institutional IACUC approval. Briefly, a dorsal midline incision was made under general anesthesia, the fallopian tubes were cauterized, both ovaries were dissected and removed, and the incision was closed with wound clips. OVX rats were shipped to our facility after full postoperative recovery.

A single subcutaneous injection of sugen (SU5416) (S8442 MilliporeSigma, CAS Number 204005-46-9, PubChem Substance ID 24278606 Sigma-Aldrich, St. Louis, MO, USA) was followed by 3 weeks of hypoxia (10% oxygen). Animals were then returned to normoxia (21% oxygen) to allow for vascular remodeling, generating different stages of PH. Based on a previous longitudinal study of right-ventricular function in male SuHx rats, Kwan et al. identified the most pronounced changes in ventricular remodeling to occur after 4 (SuHx Week 4) and 8 (SuHx Week 8) weeks post-SuHx induction [13], potentially describing an early and advanced stage of PAH. Rats kept under normoxic conditions served as controls.

2.2. Pulmonary Artery Hemodynamics

To confirm PH induction, open-chest procedures were performed at 4 or 8 weeks post-SuHx, as previously described [13,14]. Briefly, animals were anesthetized with 2.5% isoflurane (MWI Veterinary Supply, Cat. No. 502017, Boise, ID, USA) administered via tracheotomy and ventilated (Model SAR-1000, CWE Inc., Ardmore, PA, USA). Following thoracotomy, a 1.9 F admittance catheter (Transonic Scisense, London, ON, Canada) was inserted into the right ventricle to record pressure–volume waveforms (LabChart Pro v8.1.30, ADInstruments Inc., Colorado Springs, CO, USA). A 1.6 F dual-pressure catheter (FTH-1615B, Transonic Scisense, ON, Canada) was then advanced past the pulmonic valve to measure pulmonary arterial pressures.

Data was sampled at 1000 Hz and synchronized in LabChart. Pulmonary vascular resistance (PVR) was calculated as end-systolic pressure divided by cardiac output. Mean pulmonary arterial pressure (mPAP) was determined from the area under the pressure waveform. After measurements, animals were exsanguinated, hearts flushed with ice-cold phosphate-buffered saline (PBS), and lungs collected for arterial isolation.

2.3. Pulmonary Artery Histology

Right pulmonary arteries were dissected from PBS-flushed lungs, opened longitudinally, and dried, as previously described [15]. Samples were fixed in 4% paraformaldehyde for 24 h on a shaker, cryosectioned at 5 µm (La Jolla Institute for Immunology), and stained with Masson’s trichrome stain. Images were captured at 20× magnification (Evos FL Auto 2, Thermo Fisher Scientific, Waltham, MA, USA) and analyzed in ImageJ (v1.54P, NIH). One representative cross-sectional image was acquired for each vessel. For each structure, a circular region of interest (ROI) was positioned such that both boundaries of the target layer were tangent to the circle; the circle diameter, converted from pixels to micrometers using the scale bar, was recorded as the thickness. Three ROIs were placed at separate locations for each structure, and the mean value was reported as the measured thickness for the vessel.

2.4. Pulmonary Arterial Adventitial Layer Isolation

Isolated PAs were cut open with the luminal side facing upward, and intimal and medial layers were removed by gentle scraping. To validate the purity of the isolated adventitia, paired elastin immunofluorescence staining was performed on (1) intact PA cross-sections and (2) the corresponding isolated adventitial layers obtained from the same vessels. Isolated adventitia exhibited elastin signal intensity and fiber organization consistent with the adventitial region of intact vessels, without evidence of medial elastic lamina structures. These validation images are provided in Supplementary Figure S1. The remaining adventitial layer was lysed in buffer RLT (#79216, Qiagen, Hilden, Germany) using a BeadBug homogenizer (Z763705, Benchmark Scientific, Sayreville, NJ, USA). RNA was extracted with the RNeasy Universal Kit (#73404, Qiagen) per the manufacturer’s protocol, dissolved in nuclease-free water, and assessed for purity and concentration using a NanoDrop Lite spectrophotometer (Thermo Scientific, Waltham, MA, USA).

2.5. Pulmonary Arterial Adventitial Fibroblast Isolation

Pulmonary arterial adventitial fibroblasts (PAAFs) were isolated from normotensive 6–8-week-old male, ovary-intact and OVX female rats. Isolated PAs underwent a 90-min enzymatic digestion in 1 mg/mL collagenase type II (#LS004176, Worthington, NJ, USA) in DMEM (D5030, Gibco, Waltham, MA, USA) at 37 °C. The resulting cell suspension was supplied with media containing 89% DMEM, 10% fetal bovine serum (#16140, Sigma Aldrich), and 1% antibiotic–antimycotic (#15240062, Gibco) and plated in T75 flasks (#25-209, Genesee Scientific, El Cajon, CA, USA). Cells were allowed to attach for 24 h, provided fresh media, and passaged at confluence.

2.6. Mechanical Stimuli Cell Experiments

In vitro stretch experiments were performed as previously described [8]. Briefly, PAAFs (≤3 passages) were seeded on collaged-coated polyacrylamide gels (Young’s modulus: ∼0.5 kPa, ∼3 kPa, or ∼10 kPa) covalently bonded to benzophenone-treated PDMS membranes (#4026144, Dow, Midland, MI, USA) [8,16]. Cells were plated at ∼200,000 cells per gel and cultured for 48 h to allow attachment. Mechanical loading was applied using a custom-designed 3D-printed stretcher calibrated to deliver a 10% equibiaxial stretch to the PDMS membrane and attached gel. Gels assigned to the stretch group underwent continuous loading for 24 h in serum-free DMEM, whereas static controls were maintained under identical conditions without stretch. Immediately after loading, cells were lysed in 0.5 mL TRIzol (#15596026, Invitrogen, Carlsbad, CA, USA) for RNA extraction. Phase separation was performed with chloroform in Phase Lock Gel tubes (#2302830, Quantabio, Beverly, MA, USA), followed by isopropanol precipitation, ethanol washing, and resuspension in nuclease-free water.

2.7. Messenger RNA Quantification

cDNA was synthesized using Protoscript First Strand cDNA synthesis kits (#E6300L, Ipswich, MA, USA). Quantitative PCR (qPCR) was performed with the KAPA SYBR FAST universal qPCR kit (#07959362001, Kapa Biosystems, Wilmington, MA, USA) on a StepOnePlus Real-Time PCR System (#4376600, Applied Biosystems, Waltham, MA, USA). 18S ribosomal RNA served as the housekeeping gene. qPCR targets (collagen type I (Col1a1), collagen type III (Col3a1), α-smooth muscle actin (Acta2), elastin (Eln), fibronectin (Fn1), and lysyl oxidase like 1 (Loxl1)) were selected based on our previous study of male-derived PAAFs, in which these genes showed robust mechanosensitive regulation [8]. Here, we extend this panel to investigate sex and hormone-dependent differences in these established mechanosensitive ECM and fibrosis-related markers. Primer sequences are listed in Tables S1 and S2.

2.8. Statistics

To analyze nonparametric datasets with multiple factors, data were evaluated using the aligned rank transform (ART) method (R package version 0.11.2), followed by two-way analysis of variance (ANOVA), Tukey HSD post hoc tests, and Cohen’s d calculation (JMP v18 Student Edition; SAS Institute) [17].

3. Results

3.1. Rise in Pulmonary Arterial Pressures and Vascular Resistance Due to SuHx

SuHx induced a two-fold increase in mPAP and vascular resistance in all groups (Figure 1). In males, mPAP increased from 16.6 ± 0.8 mmHg to 33.6 ± 2.5 mmHg by Week 4 and 39.0 ± 4.9 mmHg by Week 8, while in intact females it increased from 13.8 ± 0.9 mmHg to 34.2 ± 2.5 and 36.0 ± 2.4 mmHg (p = 0.0002). OVX females showed an increase from 16.3 ± 1.2 mmHg to 27.6 ± 2.0 and 45.2 ± 2.6 mmHg, respectively. Similarly, vascular resistance increased from 42.5 ± 2.5 to 105.1 ± 7.3 and 117.6 ± 14.1 dyn·s·cm⁻⁵ in males, from 33.4 ± 2.0 to 77.9 ± 5.0 and 95.4 ± 14.4 dyn·s·cm⁻⁵ in intact females, and from 44.0 ± 2.8 to 100.7 ± 23.8 and 125.6 ± 13.9 dyn·s·cm⁻⁵ in OVX females. Intact females showed significantly lower vascular resistance than both male and OVX female groups. For all groups, both mPAP and vascular resistance were elevated by SuHx Week 4 and increased further by SuHx Week 8.

Figure 1.

Mean pulmonary artery pressure (A) and pulmonary vascular resistance (B) indicate significant vascular remodeling in SuHx animals relative to normotensive controls. Measurements were taken in normotensive (control, striped) and hypertensive (SuHx, solid) male (grey), intact female (blue), and OVX female (purple) rats. The most pronounced changes were observed at 4 weeks following SuHx induction. Data are shown as mean ± standard error. Individual points denote biological replicates. *** p < 0.001, **** p < 0.0001 indicates a significant difference between disease groups; # p < 0.05 indicates a significant difference between sex groups detected by Tukey HSD post hoc comparisons; ns = not significant.

3.2. PA Thickening and Fibrosis Due to SuHx

SuHx resulted in marked pulmonary arterial wall thickening across all groups (Figure 2A,B). Wall thickness roughly doubled from baseline in males and intact females, and increased by a similar magnitude in OVX females by Week 8. The collagen-rich adventitial layer also significantly thickened in all groups; however, the increase was substantially smaller in intact females compared with males (Figure 2A,C). Beyond Week 4, adventitial thickening continued in males and OVX females, while a decrease in thickness was observed in the intact females by Week 8. Intact females have significantly lower adventitia-to-vessel wall ratio than males (p = 0.02, Cohen’s d = 0.93) (Figure 2A,D). For adventitial thickness and the adventitia-to-whole-wall-thickness ratio, ART-ANOVA revealed significant interaction between sex and SuHx stages (p = 0.01) (Figure 2A,C,D). Contributing factors include the relatively modest adventitial thickening in OVX females at SuHx Week 4 and the apparent thinning of the vessel wall and adventitia in intact females by SuHx Week 8.

Figure 2.

Pulmonary artery cross-sectional histology indicates significant vascular thickening and fibrosis in SuHx animals. Masson’s trichrome-stained PA cross-sections (A) (blue = collagen; red = cytoplasm) reveal increased overall wall thickness (B) and adventitial layer thickness (C) in male, intact female, and ovariectomized (OVX) female rats, with differential changes in the adventitia: whole-vessel thickness ratio (D). Fibrotic adventitial thickening is significantly greater in males than in intact females. Data are presented as mean ± standard error of the mean. Individual points denote biological replicates. * p < 0.05, **** p < 0.0001 indicate SuHx effect compared to sex-matched controls; # p < 0.05, ## p < 0.01 denote differences between sex groups detected by Tukey HSD post hoc comparisons; ns = not significant.

3.3. PA Adventitial Profibrotic Expressions Due to SuHx

SuHx induction significantly increased adventitial expression of several profibrotic genes, including Col1a1, Col3a1, and Loxl1 (Figure 3A,B,F). For Col1a1, expression increased by up to seven-fold by Week 4 in all sex groups. In males and OVX females, expression continued to rise through Week 8, whereas intact females’ expression level declined from the group’s Week 4 values (Figure 3A). No significant SuHx-induced changes were observed for Acta2, Eln, or Fn1 (Figure 3C–E). OVX female animals exhibited significantly higher Acta2 expression level than intact ones, especially for SuHx groups (p = 0.02, Cohen’s d = 0.75). No significant statistical interaction between the factors of sex and disease stage was detected for any of the tested genes.

Figure 3.

qPCR results for Col1a1 (A), Col1a3 (B), Acta2 (C), Eln (D), Fn1 (E) and Loxl1 (F) mRNA expression in the pulmonary artery adventitial layer of SuHx animals. Significant increases in expression are observed for Col1a1, Col3a1, and Loxl1. OVX female rats show significantly greater Acta2 expression than intact ones. No significant disease stage-induced changes are detected for Eln, Fn1, or Acta2. Individual points denote biological replicates. *** p < 0.001, **** p < 0.0001 denote differences between disease groups; # p < 0.05 denotes significant differences between sex groups; ns = not significant.

3.4. Mechanical Stimuli-Induced PAAF Differential Profibrotic Gene Expression

Differential upregulation of profibrotic genes was observed in PAAFs from the three animal groups in response to mechanical stimuli (Figure 4A–G). When cultured on medium-stiffness substrates (∼3 kPa) compared with soft substrates (∼0.5 kPa); male-derived PAAFs showed significant increases in Col3a1; Loxl1; Fn1; and Acta2 (Figure 4B,C,E–G). Intact female-derived cells exhibited increased expression only for Loxl1; while OVX female-derived cells showed a significant rise only in Eln (Figure 4C,D,G). On high-stiffness substrates (∼10 kPa); intact female-derived PAAFs showed significant increases in Col1a1; Col3a1; Loxl1; Eln; and Fn1 (Figure 4A–E,G). Male-derived cells were upregulated for all tested genes, while OVX female-derived cells showed a significant increase only in Col1a1 (Figure 4A–G).

Figure 4.

qPCR results for Col1a1 (A), Col1a3 (B), Loxl1 (C), Eln (D), Fn1 (E) and Acta2 (F) mRNA expression in PAAFs cultured on substrates of varying stiffness and stretch. PAAF from male (grey), intact female (blue), and OVX female (purple) rats were maintained under static (striped) or stretched (solid) conditions. Distinct stiffness activation thresholds were observed for male (medium stiffness) and female (high stiffness) PAAFs. Data are presented as -∆∆C_T values normalized to the corresponding soft/static condition with within each sex. Individual points denote biological replicates. ART-ANOVA with Tukey HSD post hoc testing (G) identified significant effects of stiffness, stretch, and their interactions. p < 0.05 denotes significant differences compared to the soft-static baseline or as indicated in post hoc comparisons.

Under the effect of 10% equiaxial stretch, male-derived PAAFs exhibited significant increases in all tested genes, whereas intact female-derived cells responded only with increased Col3a1 expression, and for OVX female ones, Acta2 only (Figure 4A–G). Significant interactions between stretch and stiffness levels were detected in male PAAFs for Col3a1 and Fn1 and in OVX cells for Eln.

4. Discussion

This study is the first to integrate stage-specific analysis of pulmonary arterial adventitial remodeling in the SuHx rat model, combining in vivo hemodynamics, histology, layer-specific transcriptional profiling, and in vitro mechanotransduction assays of pulmonary artery adventitial fibroblasts (PAAFs) across sex and hormonal groups. For this study, we focused on the tissue- and cellular-level changes in the proximal pulmonary arteries, which undergo substantial pathological remodeling and influences right-ventricular loading and hemodynamic performance. Clinical studies and our previous work in the male rat PAH model demonstrated significant proximal PA thickening and mechanical stiffening [15,18,19]. While fibroblast phenotypes differ between large and distal vessels, our goal here was to define mechanobiologic mechanisms specific to the proximal vasculature, which plays an important role in right ventricle afterload.

Despite similar hemodynamic severity and general vascular remodeling—comparable elevations in pulmonary artery pressure and wall thickening—male, intact female, and OVX female rats exhibited distinct patterns of pulmonary vascular resistance shifts and adventitial fibrosis, both in magnitude and temporal progression. By incorporating intact and OVX females, we disentangled the contributions of ovarian hormones from chromosomal sex, revealing that hormonal status and genetic sex independently and interactively shape fibroblast mechanosensitivity and profibrotic gene expression. We also identified a previously unrecognized high stiffness activation threshold in intact female PAAFs that may prevent entry into the “stiffness → fibrosis → stiffness” feedback loop, and we report adventitial ECM gene upregulation in the absence of classical myofibroblast activation, suggesting alternative profibrotic mechanisms in PAH.

4.1. SuHx Rat Model and Hemodynamic Outcomes

The SuHx model replicates key features of human PAH—including vascular proliferation, concentric remodeling, and plexiform lesions—while supporting substantial longevity for multi-stage assessment [20,21,22]. Unlike monocrotaline, which fails to reliably induce PH in female animals [23], the SuHx model produces robust PAH phenotypes in both sexes and in OVX animals. Our previously published study demonstrated that, in addition to mean pulmonary arterial pressure and pulmonary vascular resistance, SuHx rats exhibit significant increases in pulmonary arterial elastance and reductions in right ventricle ejection fraction across all sex groups, indicating impaired RV-PA coupling [14]. These findings highlight the functional consequences of proximal pulmonary arterial remodeling, including adventitial thickening.

In our cohort, SuHx produced sustained increases in mean pulmonary arterial pressure and vascular resistance in all groups, consistent with prior reports [13,14,20,21]. The absence of significant sex differences in the mean pulmonary arterial pressure indicates that the divergent fibrosis patterns we observed are not secondary to differences in overall disease severity. This distinction is important because it isolates sex and hormonal status as independent modulators of pulmonary arterial remodeling, rather than downstream consequences of a more severe pressure load. The intact female group showed significantly lower pulmonary vascular resistance than both male and OVX female animals, with the most distinct differences observed among the SuHx groups. These comparisons indicate sex differences with ovarian hormone-dependent mechanisms during the vascular pathogenesis process, leading to pronounced discrepancies in the resulting pulmonary arterial functions.

4.2. Adventitial Remodeling and Profibrotic Gene Expression

Histology showed that all groups developed significant vascular wall thickening. Beyond the magnitude of thickening, disease progression patterns differ markedly between sex groups. Males showed consistently aggressive adventitial thickening. Both intact and OVX females exhibited less extensive fibrosis, but with distinct temporal profiles: OVX females had the least adventitial thickening at 4 weeks post SuHx induction yet continued to worsen by Week 8, whereas intact females displayed a comparable initial thickening followed by partial regression by Week 8. These patterns suggest that ovarian hormones may simultaneously contribute to early remodeling and facilitate later fibrosis resolution or remodeling reversal. In contrast, their absence appears to blunt early thickening but allows a more monotonic, progressive worsening. While speculative, this interpretation aligns with clinical observations of higher rate of PAH occurrence but better long-term prognosis in premenopausal women and warrants further mechanistic investigation.

Adventitial layer-specific gene expression analysis revealed parallel sex-dependent trends. Collagen type I, collagen type III, and lysyl oxidase like 1 (Loxl1) were significantly upregulated in males and OVX females by Week 4, with levels maintained or elevated at Week 8. In intact females, collagen type expression declined by Week 8, and Loxl1 showed much less pronounced increase, suggesting ovarian hormones attenuate late-stage profibrotic expression and may partially restore baseline transcription. The early collagen upregulation corresponded with histologic thickening, consistent with the adventitia’s central role in ECM-driven remodeling and with reported five-fold stiffness increase in SuHx arteries [7].

Our qPCR data, corroborated by αSMA immunofluorescence imaging (Supplementary Figure S2), indicate no significant SuHx-induced upregulation of Acta2 in the PA adventitia despite significant differences between intact and OVX female animals. These findings suggest that SuHx-induced adventitial fibrosis may arise through mechanisms largely independent of classical myofibroblast activation, reflecting an alternative activation state in PAAFs [4]. Notably, Loxl1 and collagen type I and III transcripts were markedly increased. Prior work, including our PAAF mechanosignaling model, has shown that YAP/TAZ activation—driven by stretch and stiffness and mediated in part by miR130/301 axis—upregulates both collagens and Loxl1 [8,24]. The expression patterns we observe are consistent with a dominant role for the YAP/TAZ pathway in driving pathological adventitial fibrosis in this SuHx model, independent of Acta2 upregulation. Quantifying the YAP/TAZ’s nuclear versus cytoplasm localization would be a promising direction for future studies. In addition, alternative fibroblast activation markers, including platelet-derived growth factor receptor alpha (PDGFRα) and fibroblast activation protein (FAP), represent promising targets for future investigation to further resolve the heterogeneity of fibroblast activation states during PAH [25,26].

4.3. Sex Differences in PAAF Mechanotransduction

In vitro mechanostimulation experiments provided mechanistic insight into these in vivo differences. Male-derived PAAFs had multiple profibrotic genes upregulated in response to both increased substrate stiffness and stretch. However, female PAAFs exhibited a markedly attenuated transcriptional response to stretch compared with males. This pattern was similar in intact and OVX groups, suggesting a role for chromosomal sex rather than circulating ovarian hormones in governing stretch sensitivity. One potential mechanism involves X-chromosome inactivation (XCI) escape genes, which are overexpressed in female cells and have been implicated in sex-biased fibroblast activation [27,28]. For example, Aguado et al. showed that XCI-escape genes such as BMX and STS modulate valvular interstitial cell responses to biochemical and mechanical cues during aortic valve stenosis [27]. We speculate that analogous X-linked regulatory pathways may blunt stretch-induced profibrotic activation in female PAAFs. Definitive identification of these mechanisms will require dedicated transcriptomic and epigenetic profiling.

The examined substrate stiffness levels were selected to mimic a physiological relevant range encompassing both healthy and diseased pulmonary arteries across PAH severity [7]. In terms of stiffness-induced upregulations, smooth muscle actin showed significant responses in male-derived cells but not in intact or OVX female-derived cells, paralleling human PAAF findings [29]. Within the female cohort, though not significantly upregulated by an increase in substrate stiffness, OVX-derived PAAFs exhibited persistent baseline elevation of elastin, fibronectin, and Loxl1 on soft substrates. Given the extended culture period, these patterns likely reflect durable transcriptional reprogramming from prior in vivo hormone loss. Notably, intact female PAAFs required a higher stiffness threshold for robust profibrotic activation. Such a threshold may delay entry into the “stiffness → fibrosis → stiffness” feedback loop, potentially contributing to slower adventitial fibrosis progression and improved prognosis in premenopausal women [9,11,12].

Our in vitro study suggests that the PAAFs derived from intact and OVX females retain distinct mechanosignaling profiles even after weeks under identical culture conditions, suggesting that ovariectomy-associated hormonal changes induce persistent transcriptional reprogramming. These long-lasting changes imply that short-term reintroduction of ovarian hormones may not fully reverse PAAF behavior. In the context of PAH, sustained hormone replacement therapy has shown both potential benefits and risks in prior studies [30]. Future work will directly assess the effects of exogenous ovarian hormone treatment on PAAFs and on vascular remodeling using controlled in vivo and in vitro paradigms.

Compared to the in vivo findings, stronger profibrotic upregulation was observed in the stiffness- and stretch-induced in vitro experiments, particularly for male-derived PAAFs and for smooth muscle actin, elastin, and fibronectin. These differences likely arise from microenvironmental factors that cannot be fully reproduced in vitro, including cell–cell crosstalk, paracrine signaling, and circulating ligands. Our system was optimized to isolate the effect of mechanical stimuli specifically, but it does not attempt to replicate the full complexity of the in vivo milieu.

4.4. Hormonal and Chromosomal Contributions to PA Remodeling

By separating intact females, OVX females, and males, we distinguished hormonal from chromosomal influences. For the in vitro experiment, hormonal status modulated PAAFs’ stiffness sensitivity, while chromosomal sex determined stretch responsiveness: only male-derived PAAFs showed significantly increased collagen type I, Loxl1, elastin, and fibronectin with stretch, regardless of hormone exposure. Again, these findings parallel evidence from valve interstitial cells that XCI escape genes can greatly alter fibroblast transcriptome and profibrotic expression [27]. Together, they reveal a dual-axis regulation—hormonal and epigenetic—of fibroblast mechanoresponses in PAH.

Overall, our findings provide a mechanistic framework for the well-described sex-differences and prognosis. We show that PAAFs from males exhibit more aggressive profibrotic responses to pathological mechanical stimuli, whereas female PAAFs—particularly in the presence of ovarian hormones—display higher thresholds for mechanosensitive activation and partial remodeling reversal. Underlying the corresponding differences in vascular fibrotic remodeling and resistance shifts, these cellular-scale differences in vascular fibrotic remodeling offer a potential explanation for why premenopausal women, despite higher PAH incidence, often experience slower disease progression and improved survival compared with men.

Given that chromosomal sex and ovarian hormone significantly influence PAH pathogenesis, existing and emerging therapies should consider sex as a critical factor when designing patient-specific treatment strategies. Therapeutically, our data highlight fibroblast mechanosensitivity and matrix remodeling as attractive targets. For example, the YAP/TAZ–Loxl1 axis implicated here suggests that LOX/LOXL inhibitors, which are already under investigation in other fibrotic diseases, could attenuate adventitial stiffening and collagen crosslinking in PAH [31,32]. Circulating ovarian hormone levels, considered alongside chromosomal sex, could clinically provide patient-specific predictors for future prognosis. In parallel, sex-informed strategies that either harness beneficial aspects of ovarian hormone signaling or mimic the protective effects of XCI-escape genes may help mitigate OVX-like reprogramming of fibroblasts [27]. Careful evaluation of menopausal hormone therapy or selective estrogen receptor modulators in PAH, with attention to fibrosis-related endpoints and corresponding early intervention, could be guided by such mechanistic insights.

4.5. Limitations

This study and corresponding experiments were designed specifically to understand the sex-dependent cellular fibrosis during PAH. Circulating ovarian hormones were not directly measured in this study. Although ovary removal in OVX animals was confirmed and is expected to substantially reduce circulating estrogen and progesterone levels [33], this quantitative comparison between intact and OVX groups—rather than direct hormone quantification—may attenuate the magnitude of observed response effects. We acknowledge that ovariectomy alters not only hormone levels but also metabolic, neural, and behavioral states. However, these systemic changes are largely secondary to the loss of ovarian hormone depletion in vivo state we aim to model. Animals were included in the study at the same age to ensure consistency, and age of the animal was not included as a factor of focus. In vitro, PAAFs were maintained in fetal bovine serum, which contains hormones and growth factors that can influence gene expression. Because all groups were cultured under identical conditions, it is unlikely that the background hormones in the medium accounted for the phenotypic differences. Culture duration was not tested as a primary variable; however, consistent results across batches confirmed that the observed sex differences were not significantly impacted by passage number (≤3) or culture length. These observations suggest that the experimental mechanical stimuli predominated over any potential effects of serum-derived hormones [34]. To recapitulate strain induced by elevated mPAP, the stretcher experiment applied sustained, equibiaxial 10% stretch for 24 h [8]. Stretch paradigms with different magnitudes, dynamics, dimensionality, or duration may produce distinct sex-specific responses in PAAFs, reflecting the heterogeneous mechanical environment of the pulmonary vasculature during PAH [7]. The in vitro substrate used in this study was a 2D hydrogel designed to allow for recapitulation of stiffness and stretch, thus unable to fully mimic the 3D in vivo environment. Although protein-level assessment (e.g., αSMA or vimentin staining) would provide valuable additional insight, our polyacrylamide-on-PDMS stretcher configuration impeded reliable fixation and imaging without disrupting cell morphology. As a result, attempted immunostaining yielded inconsistent data and was not included. Future studies using imaging-compatible platforms will be needed to pair transcriptional profiling with direct protein-level and structural assessments of cytoskeletal remodeling. This study focused solely on the pulmonary artery adventitial layer and its resident fibroblasts. Although medial and intimal layers also contribute to vascular remodeling, their roles were beyond the scope of our current design [5]. Future work incorporating whole-vessel transcriptomic or layer-specific sequencing will be essential to evaluate cell–cell crosstalk and integrated remodeling across the PA wall, while measurements such as time-lapse contractility and migration tracking and cell force quantification will provide cellular response characterization outside of gene expression.

5. Conclusions

SuHx-induced PAH produces equivalent pulmonary arterial pressure burden across sexes, yet adventitial fibrosis is profoundly modulated by ovarian hormones and chromosomal sex. Intact females exhibit attenuated vascular resistance rise, transient collagen overexpression, higher fibroblast stiffness activation thresholds, and limited stretch responsiveness, whereas OVX females display male-like remodeling kinetics despite modest stretch-induced fibrosis similar to intact ones. These findings identify a hormone-modulated stiffness threshold in female PAAFs, document persistent transcriptional reprogramming after hormone loss, and reveal chromosome-linked differences in stretch sensitivity. The absence of myofibroblast marker induction despite robust ECM gene activation challenges canonical models of fibrosis initiation, pointing to alternative PAAF-driven pathways. Collectively, these advances provide new mechanistic insight into sex-specific vascular remodeling in PAH and suggest that modulating adventitial fibroblast mechanosensitivity could offer a stage- and sex-tailored therapeutic strategy.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cells15040354/s1.

Author Contributions

Y.L., A.W. and D.V.-J. conceived and designed research; Y.L., A.W. and D.V.-J. performed experiments; Y.L., A.W. and D.V.-J. analyzed data; Y.L. and D.V.-J. interpreted results of experiments; Y.L., A.W. and D.V.-J. prepared figures; Y.L. drafted manuscript; D.V.-J. edited and revised manuscript; Y.L., A.W. and D.V.-J. approved final version of the manuscript. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The animal study protocol was approved by the Institutional Animal Care and Use Committee at the University of California, San Diego (Protocol Code: S17237; Date of Approval: 30 January 2024).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

No conflicts of interest, financial or otherwise, are declared by the authors.

Funding Statement

This work was funded in part by American Heart Association Grant 16SDG29670010 (to D.V.-J.) and 25PRE1363629 (to Y.L.); National Heart, Lung, and Blood Institute of the National Institutes of Health under Grants R25HL145817, R01HL155945 (to D.V.-J.), T32HL160507 (to Y.L.), 1T32HL105373 (to A.W.); National Science Foundation Grant CAREER 2046259 (to D.V.-J.); the Conrad Prebys Foundation (to D.V.-J.); and the Wu-Tsai Foundation (to D.V.-J.).