Gregory N. Adams

Murine prolylcarboxypeptidase depletion induces vascular dysfunction with hypertension and faster arterial thrombosis

Prolylcarboxypeptidase (PRCP) activates prekallikrein to plasma kallikrein, leading to bradykinin liberation, and degrades angiotensin II. We now identify PRCP as a regulator of blood vessel homeostasis. β-Galactosidase staining in PRCP(gt/gt) mice reveals expression in kidney and vasculature. Invasive telemetric monitorings show that PRCP(gt/gt) mice have significantly elevated blood pressure. PRCP(gt/gt) mice demonstrate shorter carotid artery occlusion times in 2 models, and their plasmas have increased thrombin generation times. Pharmacologic inhibition of PRCP with Z-Pro-Prolinal or plasma kallikrein with soybean trypsin inhibitor, Pro-Phe-Arg-chloromethylketone or PKSI 527 also shortens carotid artery occlusion times. Aortic and renal tissues have uncoupled eNOS and increased reactive oxygen species (ROS) in PRCP(gt/gt) mice as detected by dihydroethidium or Amplex Red fluorescence or lucigenin luminescence. The importance of ROS is evidenced by the fact that treatment of PRCP(gt/gt) mice with antioxidants (mitoTEMPO, apocynin, Tempol) abrogates the hypertensive, prothrombotic phenotype. Mechanistically, our studies reveal that PRCP(gt/gt) aortas express reduced levels of Kruppel-like factors 2 and 4, thrombomodulin, and eNOS mRNA, suggesting endothelial cell dysfunction. Further, PRCP siRNA treatment of endothelial cells shows increased ROS and uncoupled eNOS and decreased protein C activation because of thrombomodulin inactivation. Collectively, our studies identify PRCP as a novel regulator of vascular ROS and homeostasis.

Prolylcarboxypeptidase promotes angiogenesis and vascular repair

Prolylcarboxypeptidase (PRCP) is associated with leanness, hypertension, and thrombosis. PRCP-depleted mice have injured vessels with reduced Kruppel-like factor (KLF)2, KLF4, endothelial nitric oxide synthase (eNOS), and thrombomodulin. Does PRCP influence vessel growth, angiogenesis, and injury repair? PRCP depletion reduced endothelial cell growth, whereas transfection of hPRCP cDNA enhanced cell proliferation. Transfection of hPRCP cDNA, or an active site mutant (hPRCPmut) rescued reduced cell growth after PRCP siRNA knockdown. PRCP-depleted cells migrated less on scratch assay and murine PRCP(gt/gt) aortic segments had reduced sprouting. Matrigel plugs in PRCP(gt/gt) mice had reduced hemoglobin content and angiogenic capillaries by platelet endothelial cell adhesion molecule (PECAM) and NG2 immunohistochemistry. Skin wounds on PRCP(gt/gt) mice had delayed closure and reepithelialization with reduced PECAM staining, but increased macrophage infiltration. After limb ischemia, PRCP(gt/gt) mice also had reduced reperfusion of the femoral artery and angiogenesis. On femoral artery wire injury, PRCP(gt/gt) mice had increased neointimal formation, CD45 staining, and Ki-67 expression. Alternatively, combined PRCP(gt/gt) and MRP-14(-/-) mice were protected from wire injury with less neointimal thickening, leukocyte infiltration, and cellular proliferation. PRCP regulates cell growth, angiogenesis, and the response to vascular injury. Combined with its known roles in blood pressure and thrombosis control, PRCP is positioned as a key regulator of vascular homeostasis.

The Williams-Beuren Syndrome-a window into genetic variants leading to the development of cardiovascular disease.

Williams-Beuren Syndrome (WBS) arises when there is a genomic microdeletion at human chromosome 7q11.23 (Mouse 5G2), resulting in various cardiovascular, developmental, metabolic, and mental disorders [1]. Cardiovascular complications from WBS are a frequent cause of death. The deleted region is predisposed to non-allelic homologous recombination (NAHR) due to the presence of repetitive DNA regions called segmental duplications. WBS can result in deletions of up to 1.83 Mb in a region containing roughly 28 genes that includes the gene ELN encoding the tissue structural protein elastin [2]. Many of the cardiovascular features of WBS can be partially explained by elastin defects. WBS individuals have a combined prevalence of cardiovascular disease of 84% that includes supraand sub-aortic stenosis (SVAS); aortic, pulmonary, and mitral valvular disease; aortic coarctation; hypertension; and, less commonly, myocardial infarction [2,3]. These defects have also been found in mouse models [4]. Elastin not only provides ‘‘elastic’’ support for vessels, it also serves as a negative regulator for smooth muscle cell proliferation. WBS patients are also at high risk for hypertension and Eln mice are hypertensive [1,4]. In 2006, Del Campo et al. recognized that hemizygosity of the NCF1 gene as a result of the largest recognized WBS microdeletion—about 1.83 Mb—decreases the risk for hypertension in WBS patients compared to those possessing the more common smaller deletion (1.55 Mb) not incorporating NCF1 (Figure 1, top) [5]. NCF1 codes for p47, a critical subunit in the assembly of NADPH oxidase (NOX); homozygous deficiency accounts for 20% of patients with chronic granulomatous disease, a disorder associated with repeated infections due to an inability to kill bacteria. p47 is a major effector of angiotensin II (AngII), as demonstrated by a lack of elevation in blood pressure of Ncf1 mice [6]. In this issue of PLoS Genetics, Campuzano and colleagues replicate the WBS cardiovascular phenotype in a WBS mouse model with and without a deletion of the Ncf1 gene [7]. Their study has broad implications for our understanding of hypertensionand reactive oxygen species (ROS)-related cardiovascular disease.