The work was published in the Journal of Clinical Investigation and comes as part of the long standing collaboration between Taylor, who studies adhesion/small GTPase signaling in the heart and vasculature, and Mack who studies the transcription mechanisms that control gene expression in smooth muscle cells (SMC).
Previous studies have shown that RhoA-dependent control of SMC contractility contributed to the regulation of blood pressure, and perhaps to the development of hypertension, but the mechanisms that controlled RhoA activity in SMC were not completely clear. The Taylor lab's identification of ArhGAP42 as Rho-specific GAP that is selectively expressed in SMC was a very important advance and their studies in genetically engineered mice revealed that depletion of ArhGAP42 led to a significant dose-dependent increase in basal blood pressure (BP) due to increased Rho-dependent SMC contractility in resistance.
There were still unanswered questions, however, including how endogenous levels of ArhGAP42 are regulated and if alterations in this gene influence the development of hypertension in human.
“We were extremely excited to discover the existence of a BP-associated locus within FLJ32810 because it suggested that ArhGAP42 may play a role in pathogenesis of human hypertension," Taylor said.
The minor allele that was associated with a decrease in blood pressure was defined by several nucleotide polymorphisms (SNPs) in high linkage disequilibrium within the non-coding 80Kb ARHGAP42 first strongly suggesting that its effects were mediated by changes in ARHGAP42 expression not ARHGAP42 protein structure.
"We were perfectly positioned to identify the transcription mechanism that regulate ARHGAP42 expression and to identify the mechanisms by which variations controlled ARHGAP42 levels in SMC,” Mack said.
To this end, the Mack lab took advantage of their genome-wide data sets on chromatin structure in SMC to identify a regulatory element encompassing the blood pressure-associated SNP, rs604723, that exhibited strong SMC-selective activity. CrispR/Cas9-mediated deletion of this regulatory element in human aortic SMC cultures significantly reduced endogenous ARHGAP42 expression, and the activity of this element was significantly enhanced by the presence of the minor T allele. They also used allele-specific RT PCR to demonstrate that the minor T allele was expressed more strongly than the major C allele in our HuAoSMCs that are heterozygous at the rs604723 SNP.
This result was in excellent agreement with data from the NIH-sponsored GTEx consortium which demonstrated that ARHGAP42 expression in human aortic, coronary, and tibial artery samples was approximately three fold higher in individuals homozygous for the minor allele than in individuals homozygous for the major allele. DNA binding and transcription assays demonstrated that the presence of the minor T allele at rs604723 created a low affinity binding site for the transcription factor serum response factor (SRF) that they had previously implicated in SMC-specific gene expression.
The Taylor lab went on to show that ARHGAP42 expression in vascular SMC was up-regulated by cell stretch, hypertension, and RhoA-dependent agonists suggesting that it may act as a negative feedback mechanism to limit excessive vessel constriction. In support of this idea, DOCA-salt-mediated hypertension was exacerbated in ARHGAP42 deficient mice.
To begin to study the clinical relevance of genetic variations in ARHGAP42, they established a collaboration with Anthony Viera, a UNC physician to genotype his well-characterized cohort of untreated borderline hypertensive patients. Results confirmed that the minor ARHGAP42 allele was associated with reduced diastolic blood pressure and genotypic analysis of approximately 1,000 individuals from several additional clinical cohorts suggested that the low frequency of the minor ARHGAP42 allele in African Americans may contribute to the susceptibility of this population to the development of hypertension.
These studies were supported by a 2.9M multi-PI grant from the NIH/NHLBI awarded to Taylor, Mack and Viera. As a research team "We are particularly excited that our work could lead to new definitions for hypertensive associated cardiovascular risk, to new therapeutic opportunities in the treatment of hypertension, and perhaps, to the development of more personalized treatment options."