Sorting Out Vasoreactivity in Pulmonary Arterial Hypertension with SNX29

Vasoreactivity in WSPH Group 1 pulmonary arterial hypertension (PAH), defined by a decrease in mean pulmonary artery pressure (mPAP) by ≥10 mmHg to a value ≤40 mmHg without a decrease in cardiac output following vasodilator challenge,^1,2 has long been held to be predictor of improved outcome in this disease.^3–5 Identifying a subset of patients who are potential long-term responders to calcium channel blockers (CCB). patients with positive acute vasodilator responses (AVR) make up 5–7% of those diagnosed with idiopathic, heritable, or drug-associated PAH, with improved survival when appropriately diagnosed and treated. Limiting the potential utility of this endotype, only half of patients initially identified as vasoreactive will remain responsive after one year, and the use of calcium channel blockers is not without risk, with dose-limiting hypotension and the risk of precipitating right ventricular failure.^2,4 Current recommendations advise repeating acute vasoreactivity testing at intervals of 3–6 months up through the first year to identify long-term responders.^1,4,6 A marker that identifies long-term responders early might not only improve treatment outcomes, but could reveal strategies for leveraging the biology of long-term responses across other endotypes in PAH.

In this issue, Karnes et al. present a genome wide association study (GWAS) of AVR in PAH, using change in mean pulmonary arterial pressure with vasodilator challenge, followed by an analysis of potential physiologic mechanisms by which the identified locus and single-nucleotide polymorphism (SNP) might contribute.⁷ The result is a a novel framework linking intracellular trafficking and calcium signaling to an important PAH endotype, potentially opening the door to methods for exploiting this framework for improved risk stratification and therapy by harnessing the biology underlying this genetic marker.

For their GWAS derivation cohort, the authors leverage the PAH Biobank (n=467, but restricted to 415 subjects of European ancestry), a well-phenotyped national repository, followed by replication in two cohorts from additional US repositories, the Vanderbilt BioVU (up to n~66 with Group 1 PAH) and the Allegheny Health Network (AHN, up to n~1198 with Group 1 PAH). Of several loci identified in the discovery cohort, only an intronic SNX29 variant rs8057488 demonstrated nominal replication, with consistent directionality across cohorts. Carriers of the minor T allele exhibited greater reductions in mPAP during vasodilator testing, suggesting a role of this locus and variant in enhanced vasoreactivity, decreased disease burden, or both. Using PHBI RNA-Seq data, they found decreased expression of SNX29 mRNA in PAH lungs versus failed donor lungs, but relatively increased expression among PAH carrying T alleles compared to those without. These observations were further supported by finding increased expression of SNX29 mRNA in circulating lymphocytes from patients with vasoreactive PAH (VR-PAH) vs. non reactive PAH (NR-PAH), as well as from with VR-PAH with C/T or T/T genotype compared to NR-PAH with the C/C genotype.

The authors explored a link between SNX29 and calcium signaling via store-operated calcium entry (SOCE), based on molecular associations of SNX29 with the localization and expression of STIM1, a sarcoplasmic calcium sensor, and ORAI1, a store-operated calcium channel that is activated by STIM1 when calcium stores are low. In pulmonary artery smooth muscle cells (PASMCs), the authors found that SNX29 localizes to early to late endosomal compartments, and when overexpressed localized with STIM1 to impair its interaction with ORAI1, suppressing SOCE (Figure). Conversely, silencing of SNX29 increased expression and membrane localization of STIM1 and ORAI1, facilitating SOCE and increasing calcium influx. Given the central role of calcium signaling in regulating vascular tone and remodeling,^8,9 these findings position SNX29 as a negative regulator of SOCE. To explore the link of SNX29 and the hypoxic vasoconstrictive response, the authors pursued two in vivo strategies: Virally-mediated overexpression of Snx29 attenuated hypoxia-induced pulmonary vasoconstriction in isolated perfused mouse lungs. However, overexpression of Snx29 did not significantly alter the development of chronic hypoxia-induced pulmonary hypertension in mice, suggesting a preferential role in acute vasomotor regulation rather than structural remodeling. Taken together, these data support a model in which SNX29 modulates pulmonary vascular tone by regulating trafficking and function of SOCE components, and provide a potential explanation for enhanced vasodilator responsiveness associated with increased expression of SNX29 due to the T allele.

Figure.

SNX29 is associated with the early to late endosomal pathway and the endoplasmic reticulum (ER), and regulates the translocation of STIM1, which senses low Ca+2 in the ER, to associate with ORAI1 in the membrane, thereby promoting STORE-operated calcium entry.

This study is notable for its focus on a rigorously defined, prognostically important phenotype of AVR as a continuous variable, in contrast to GWAS and WES approaches to PAH using the presence of PAH, IPAH, or vasoreactivity as dichotomous variables, affording potentially greater statistical power for this question.^10,11 This study also demonstrates the feasibility of leveraging national biorepositories with deeply phenotyped populations to perform a GWAS analysis to yield a significant signal despite the rarity of the disease. The authors go further to pursue mechanistic validation, using human tissue and gene expression data, in vitro analysis in a relevant cell type, and experimental models, strengthening the validity of the GWAS signal to link SNX29 to calcium signaling and vascular tone. Their findings begin to clarify the mechanism that makes certain patients with PAH responders to calcium channel blockers, paving the way for further investigation of the pathway and possible new therapeutic targets.

However, several limitations warrant careful consideration. While larger than previous efforts probing this question, the modest size of the discovery and replication cohorts remains a key constraint. While identification of a genome-wide significant association is noteworthy, replication of the SNX29 signal in the validation cohort is nominal, suggesting the need for additional validation in larger cohorts. The authors mitigate these issues with the consistent directionality of this signal in both cohorts, and support from gene expression and mechanistic studies, but there remain important disconnects. The human gene expression studies show clearly that VR-PAH, and VR-PAH T allele carriers express higher levels of SNX29 in lymphocytes than NR-PAH and NR-PAH without T alleles, respectively, demonstrating that SNX29 expression is positively associated with enhanced vasoreactivity. The mechanistic experiments are consistent in demonstrating that elevated SNX29 expression suppresses SOCE membrane localization and signaling, while ex vivo perfused lung studies demonstrate increased SNX29 expression suppresses acute hypoxic vasoconstriction, suggesting SNX29 suppresses acute vasoreactivity. However, sensitivity to vasodilator may not map directly onto hypoxic vasoconstriction or SOCE function and conceivably could have inverse relationships. Similarly, PAH patients as a whole have decreased SNX29 expression in their lungs compared to failed donors, suggesting SNX29 expression could be modulated by negative feedback in the presence of high pressures. While the frequency of the T allele was enriched in VR-PAH patients across the cohorts (0.107), it was not absent in NR-PAH patients (0.052), suggesting additional factors contribute to vasoreactivity. The authors acknowledge that the molecular mechanisms linking SNX29 to SOCE require further investigation, e.g., to discern the precise nature of the SNX29 and STIM1 interaction, and the role of its phosphatidylinositol binding PX domain as seen with other SNX family members. Finally, the derivation cohort was restricted to individuals of European ancestry, limiting replication in a more diverse validation cohort given the higher frequency of rs8057488 in non-Europeans (~0.20–030 vs. 0.5), as well as limiting sensitivity for genes in non-Europeans.

The translational relevance of the in vitro and in vivo models also deserves consideration. The expression of SNX29 was enhanced in PAH T allele carriers in whole lungs and lymphocytes, while mechanistic studies were done in isolated PASMC. Understanding the impact of the rs8057488 on SNX29 expression in PASMC and other vascular lineages in clinical PAH tissues would help to validate this concept further. Hypoxia-induced pulmonary hypertension does not recapitulate the pathobiology of Group 1 PAH, and acute hypoxic vasoconstriction while known to be SOCE driven, has no necessary relationship to vasoreactivity in PAH. The exclusive use of male animals, while a common practice in experimental pulmonary hypertension studies, limits assessment of sex-specific effects, known to be an important overlay to vasoreactivity in PAH populations.^12,13 And lastly, the implications for clinical practice warrant additional longitudinal studies. While this variant appears to predict AVR, it is not clear if it this variant will also be associated with long-term response to calcium channel blockers, and ultimately the basis for the long-term survival phenotype that is being sought in these studies.