David A. Tulis, PhD - US grants

Vascular smooth muscle (VSM) cyclic guanosine 3',5'-moriophosphate (cGMP) serves as a critical[unreadable] regulator of many cellular functions that contribute to vessel growth after injury. Nitric oxide (NO) and[unreadable] carbon monoxide (CO) operate as soluble guanylate cyclase (sGC)-activating ligands for cGMP synthesis;[unreadable] however, limitations of NO and CO signaling warrant study into alternate, pathophysiologically relevant[unreadable] routes for cGMP control. Provocative new findings challenge the traditional notion that cGMP exerts[unreadable] vascular protection through cGMP-dependent protein kinase type (cGKI) and suggest that cGMP may[unreadable] operate via cAMP/cAK to promote vascular protection. Current studies in our laboratory focus on novel NOindependent[unreadable] approaches for cGMP control as significant basic science tools and as potential cardiovascular[unreadable] therapeutics. Preliminary data support a role for vascular growth control by NO-independent cGMP and[unreadable] suggest mechanistic involvement of matrix metalloproteinase (MMP)-2 and MMP-9. The long-term objective[unreadable] of this research project is to investigate strategies for cGMP control of VSM growth, and the central[unreadable] hypothesis of this proposal is that NO-independent cGMP protects against vascular growth and that this[unreadable] occurs through cAK signals. Two Specific Aims will be used to test this hypothesis:[unreadable] Aim 1 will analyze the roles of NO-independent cGMP and cGMP-directed cGKI/cAK signaling in[unreadable] attenuating vascular remodeling in the rat balloon injury and mouse wire denudation injury models.[unreadable] Aim 2 will examine matrix-based mechanisms including cell migration and MMP balance that underlie[unreadable] cGMP-mediated growth control in rat and mouse primary VSM cells.[unreadable] Pharmacology, RNA interference, and viral gene delivery approaches will be used, and conditional VSMspecific[unreadable] cGKI-deficient models will allow direct comparison of cGKI versus cAK mechanisms. Results are[unreadable] anticipated to provide insight into and further evidence for NO-independent cGMP control of the injury[unreadable] growth response in VSM and shed light upon cGMP-directed MMPs in mediating these events.[unreadable] Injuries and diseases of the heart and blood vessels are wide-ranging and very serious public health[unreadable] concerns, and statistics show they are still the major cause of death in American populations. We believe[unreadable] that results from these studies will shed light on some novel and promising strategies that could be used to[unreadable] minimize the severity of blood vessel injury and disease and may offer beneficial prospects for further study[unreadable] in basic science research and human-based clinical studies.[unreadable]

PROJECT SUMMARY/ABSTRACT Cardiovascular disease (CVD) remains the primary cause of morbidity and mortality in the United States and worldwide, and key underpinnings in CVD pathogenesis include vascular endothelial cell (VEC) inflammation and adhesion and abnormal growth of vascular smooth muscle (VSM). In diseased tissue the local microenvironment becomes acidic from altered cellular metabolism and compromised blood flow, yet the exact contributions of acidic pH to the disease process and in particular, to VEC and VSM dysfunction, is potentially significant yet not well understood. Intriguingly, a family of pH-sensing G protein-coupled receptors (GPCRs) has been identified including GPR4, primarily found in VECs, and GPR68, predominantly localized to VSM, and recent findings suggest these may be crucial in eliciting VEC and VSM complications foundational to CVD. The broad goal of this research plan is to determine precise roles and mechanisms of GPR4 and GPR68 in soliciting pathologic VEC inflammation and adhesion and VSM growth. This line of study directly addresses the health concerns of CVD and is of potential clinical importance. The hypothesis of this project is that acidosis activates pH-sensing VEC GPR4 and VSM GPR68, thereby stimulating cyclic AMP-driven Epac and inhibiting anti-inflammatory and growth-protective AMPK, in turn promoting VEC inflammation and adhesion and deleterious VSM growth as foundations of vascular dysfunction in CVD. Using wild type (WT), GPR4 knockout (KO) and GPR68 KO mice and in vitro and in vivo approaches with gain-of-function/loss-of-function interventions to validate mechanisms, three Specific Aims will test our hypothesis: Aim 1 will examine cellular signals in response to acidosis including cyclic AMP content, activities of cyclic AMP-dependent protein kinase (PKA) and cyclic AMP-degrading phosphodiesterase (PDE), and expression and activities of downstream effectors Epac and metabolic AMP-dependent protein kinase (AMPK). Aim 2 will determine the regulatory impacts of GPR4 and GPR68 signals on VEC inflammation and adhesion and VSM cell (VSMC) migration and proliferation, and Aim 3 identify discrete GPR4 or GPR68 processes capable of controlling arterial growth and remodeling under in vivo conditions. This integrated research design will determine pH-sensing GPR4 and GPR68 and their intracellular effectors Epac and AMPK as instrumental in VEC inflammation and adhesion and VSM migration and proliferation elemental to CVD. Anticipated findings promise to shift our current understanding of vascular cell signaling and will provide new avenues for basic and clinical investigation with the hopes of identifying novel, more selective targets for therapeutic intervention in CVD patients.