Pulmonary Hypertension: Beyond Vasodilators

Sustained pulmonary vasoconstriction, concentric pulmonary vascular remodeling, obliterative pulmonary vascular lesions and pulmonary vascular wall stiffening are the major causes for the elevated pulmonary vascular resistance (PVR) in patients with pulmonary arterial hypertension (PAH) and other forms of pre-capillary pulmonary hypertension (PH). An increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) due to Ca²⁺ influx through cation channels in the plasma membrane of pulmonary arterial smooth muscle cells (PASMCs) triggers PASMC contraction inducing pulmonary vasoconstriction and stimulates PASMC proliferation resulting pulmonary vascular wall thickening. Transient receptor potential canonical (TRPC) channels are a family of non-selective cation channels involved in mediating receptor-operated Ca²⁺ entry (ROC) that induces vascular SMC contraction, migration and proliferation. TRPC6, a TRPC channel that forms ROC in vascular SMC including PASMCs, is activated by diacylglycerol (DAG), an important second messenger for multiple membrane receptors. We and others have observed that TRPC6, along with other cation channels, is upregulated in PASMCs from patients with PAH and animals with experimental PH. In this study, we report that oral administration of BI-749327, a selective blocker of TRPC6 channels (IC₅₀ = 13-19 nM), partially reversed the established experimental PH in animals. The therapeutic or reversal effect of BI-749327 was associated with its inhibitory effect on the Ca²⁺-dependent activation of AKT/mTOR signaling pathway in PASMCs. Acute treatment of isolated and perfused/ventilated lungs with BI-749327 also inhibited alveolar hypoxia-mediated pulmonary vasoconstriction. These data indicate that inhibition of TRPC6 channels is potentially a novel and effective therapeutic approach for PAH and other forms of pre-capillary PH.

Pulmonary arterial hypertension (PAH) is deadly disease with no cure, characterized by pulmonary vascular remodeling, increased pulmonary artery (PA) pressure and right ventricle failure. We previously reported that tuberous sclerosis complex 2 (TSC2) acts as a mechanosensor and mechanotransducer, and its deficiency in PA vascular smooth muscle cells (PAVSMC) promotes PAVSMC hyper-proliferation through YAP-mTOR, which is suppressed by restoration of functional TSC2 by SRT2104. The role of TSC2 deficiency in PAH/PH development and whether pharmacological TSC2 restoration reverses severe PH remain undetermined. We here report that extracellular matrix, produced by TSC2-deficient PAVSMC, up-regulates YAP-mTOR and promotes PAVSMC growth and proliferation, suggesting that TSC2 acts via modulation of ECM. Compared to same age/sex controls, SMC-specific heterozygousTsc2 deletion leads to spontaneous PH in 9 weeks-old male and four out of six female mice. Further, oral administration of SRT2104 to rats with SuHx-induced PH restored SMC Tsc2 in small PAs, reversed pulmonary vascular remodeling, decreased percentage of occluded PAs, and resolved PH. In conclusion, TSC2 deficiency in PAVSMC induces early onset of spontaneous PH in mice. SRT2104 restores Tsc2 in small PAs and reverses pulmonary vascular remodeling and severe experimental PH in rats.

Pulmonary hypertension (PH)-induced RV failure (PH-RVF) is characterized by EndMT and fibrosis. The link between RV-EndMT and fibrosis and their regulation is yet to be elucidated. Hypothesis: Snai1 regulates RV-fibrosis via LOXL2-mediated mechanism. Snai1 knockdown (KD) may serve as potent RV-specific therapy to rescue PH-RVF via inhibiting RV-EndMT and fibrosis. Methods: MCT and SuHx rats (n=9) received siSnai1 (5nM every 3-4d, IV) or scramble (n=10) as rescue. Echo and cath, RV-RNASeq, qPCR and WB validation were performed. Rat RVs were stained for EndMT (CD31+αSMA) and fibrosis (trichrome; Vimentin+αSMA) and Snai1+LOXL2. Snai1-KD was performed on human coronary artery endothelial cells (HCAECs) under hypoxia+TGFβ1(72h). Further, human cardiac fibroblasts (HCFs) were cultured with hypoxia+TGFβ1(48h) or HCAEC-conditioned medium(72h)±LOXL2 inhibitor. Results: Snai1-KD resulted in decreased RV-Snai1 expression, RVSP, FI and increased RVFAC. Snai1-KD also resulted in decreased RV-LOXL2, EndMT and fibrosis. RV-RNASeq showed EndMT as the top downregulated pathway and decreased fibrotic DEGs. Snai1-KD inhibited hypoxia+TGFβ1-induced EndMT in HCAECs by decreasing Snai1+LOXL2 expression and co-localization. Both hypoxia+TGFβ1 and HCAEC-conditioned medium induced CF-myofibroblast transition that was prevented by LOXL2 inhibition. Conclusions: Targeting Snai1 is a novel RV-specific therapy for PH-RVF via LOXL2-mediated inhibition of EndMT and fibrosis.

Pulmonary arterial hypertension (PAH) is characterized by vascular remodeling and vascular resistance, which may lead to maladaptive right ventricular (RV) failure and ultimately death. Our previous studies showed that intratracheal delivery of AAV1.SERCA2a inhibited vascular remodeling and restored RV function in the monocrotaline-induced PAH model. However, the molecular mechanisms by which SERCA2a regulates PASMC proliferation remain poorly understood. RESULTS: Here, we found that decreased SERCA2a expression in PAH patients' lung samples was associated with downregulation of BMPR2 and activation of STAT3. Analysis of the transcriptome profile by RNA-sequencing of PASMCs co-overexpressing SERCA2a and BMPR2 identified STAT3 among the top regulated transcription factors. Using a specific siRNA against STAT3 and a pharmacological STAT3 inhibitor (HJC0152), we found that SERCA2a potentiated BMPR2 expression by repressing STAT3 activity. In vivo, we found that combined therapy of AAV1.hSERCA2a with AAV1.hBMPR2 or HJC0152 enhanced SERCA2a’s beneficial effects. Using cardiac magnetic resonance imaging, we found that therapies using AAV1.hSERCA2a alone or combined with HJC0152 significantly inhibited RV structural and functional changes in a severe model of PAH. CONCLUSIONS: Our study demonstrated that combination therapies using SERCA2a-gene transfer with a pharmacological inhibitor of STAT3 could represent a new promising therapeutic alternative to reverse PAH.

With severe right ventricular (RV) pressure overload, women demonstrate better clinical outcomes compared to men. The mechanoenergetic mechanisms underlying this protective effect, and their dependence on female endogenous sex hormones, remain unknown. To investigate these mechanisms and their impact on RV systolic and diastolic functional adaptation, we created comparable pressure overload via pulmonary artery banding (PAB) in intact male and female Wistar rats and ovariectomized females. At 8 weeks post-surgery, right heart catheterization demonstrated increased RV energy input (indexed pressure-volume area) in all PAB groups, with the greatest increase in intact females. PAB also increased RV energy output (indexed stroke or external work) in all groups, again with the greatest increase in intact females. In contrast, PAB increased RV contractility (indexed end systolic elastance) in females but not males. Despite these sex-dependent differences, the decrease in ratio of RV energy output to input (mechanical efficiency) and increase in mechanoenergetic cost to pump blood with pressure overload were not sex-dependent. These metrics were similarly unaffected by loss of endogenous sex hormones in females. Finally, despite sex-dependent differences in collagen content and organization with pressure overload, decreases in RV compliance and relaxation time constant (Tau Weiss) were not sex-dependent. In sum, despite sex-dependent differences in RV contractile and fibrotic responses, RV mechanoenergetics for this degree and duration of pressure overload are comparable between sexes and suggest a homeostatic target.