Anti-PRKCD (aa 577-676) polyclonal antibody

Rationale: Regeneration of denuded or injured endothelium is an important component of vascular injury response. Cell-cell communication between endothelial cells and smooth muscle cells (SMCs) plays a critical role not only in vascular homeostasis but also in disease. We have previously demonstrated that PKCd (protein kinase C-delta) regulates multiple components of vascular injury response including apoptosis of SMCs and production of chemokines, thus is an attractive candidate for a role in SMC-endothelial cells communication. Objective: To test whether PKCd-mediated paracrine functions of SMCs influence reendothelialization in rodent models of arterial injury. Methods and Results: Femoral artery wire injury was performed in SMC-conditional Prkcd knockout mice, and carotid angioplasty was conducted in rats receiving transient Prkcd knockdown or overexpression. SMC-specific knockout of Prkcd impaired reendothelialization, reflected by a smaller Evans blue-excluding area in the knockout compared with the wild-type controls. A similar impediment to reendothelialization was observed in rats with SMC-specific knockdown of Prkcd. In contrast, SMC-specific gene transfer of Prkcd accelerated reendothelialization. In vitro, medium conditioned by AdPKCd-infected SMCs increased endothelial wound closure without affecting their proliferation. A polymerase chain reaction-based array analysis identified Cxcl1 and Cxcl7 among others as PKCd-mediated chemokines produced by SMCs. Mechanistically, we postulated that PKCd regulates Cxcl7 expression through STAT3 (signal transducer and activator of transcription 3) as knockdown of STAT3 abolished Cxcl7 expression. The role of CXCL7 in SMC-endothelial cells communication was demonstrated by blocking CXCL7 or its receptor CXCR2, both significantly inhibited endothelial wound closure. Furthermore, insertion of a Cxcl7 cDNA in the lentiviral vector that carries a Prkcd shRNA overcame the adverse effects of Prkcd knockdown on reendothelialization. Conclusions: SMCs promote reendothelialization in a PKCd-dependent paracrine mechanism, likely through CXCL7-mediated recruitment of endothelial cells from uninjured endothelium.

We have previously reported that casein hydrolysate, CH-3, from bovine milk and casein-derived tripeptide Met-Lys-Pro (MKP) has ACE inhibitory activity and reduces blood pressure. In this study, we investigated the therapeutic effects of MKP in a hypertensive rat model (7-week-old male SHRSP/Izm rats). For long term evaluation, rats were fed either a diet containing CH-3 or normal diet. The survival rate of SHRSP rats was significantly improved by intake of CH-3 for 181 days. For short term evaluation, rats were orally administered synthetic tripeptide MKP or distilled water for 4 weeks. MRI study demonstrated that hemorrhagic lesions were observed in two of five rats in the control group, while no hemorrhagic lesions were observed in the MKP group. Volumetric analysis using MRI revealed that MKP administration inhibited atrophy of diencephalic regions. Histological examinations revealed that hemorrhage areas and astrogliosis in the hippocampus and cerebral cortex were lower in the MKP group than in the control group. Gene expression analysis indicated that MKP administration reduced expression of genes related to cerebral circulation insufficiency such as immune responses (Cd74andPrkcd), response to hypoxia (Ddit4,Apold1, andPrkcd), reactive oxygen species metabolic process (Ddit4andPdk4), and apoptotic process (Ddit4,Prkcd, andSgk1), suggesting that MKP administration prevented cerebral ischemia associated with hypertension. In addition, some genes encoding responses to hormone stimulus (Fos,Dusp1, andSik1) were also downregulated. Serum aldosterone and corticosterone levels were also significantly decreased following MKP administration. The present study indicates that MKP shows neuroprotective effects in SHRSP rats by regulating cerebral circulation insufficiency and corticoid levels. MKP administration may therefore be a potential therapeutic strategy for hypertensive brain diseases such as cerebrovascular disease.