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Vascular calcification results from deposition of calcium hydroxyapatite crystals in the vessel wall. It is highly prevalent in patients with chronic kidney disease (CKD), diabetes, and peripheral artery disease (PAD). In lower extremity arteries, elevated calcification levels are associated with an increased risk of ischemic events including amputation. The cyclic nucleotides cAMP and cGMP, controlled by distinct cyclic nucleotide phosphodiesterase (PDE) isozymes, play important regulatory roles in a variety of human diseases. Using a qPCR PDE array, we found that PDE10A was the most highly induced isoform in a rodent model of arterial calcification. PDE10A was also markedly increased in calcifying vascular smooth muscle cells (VSMCs) in vitro , calcified arteries from rodents with CKD, and calcified human tibial arteries. Knockdown or inhibition of PDE10A markedly attenuated high phosphate-induced VSMC osteogenic transformation and calcification in both rat and human VSMCs. Importantly, deficiency of PDE10A significantly decreased arterial calcification in both ex vivo aortic ring and in vivo vitamin D 3 medial calcification models. Several labs including our own have previously demonstrated that matrix metallopeptidases (MMPs) are involved in vascular calcification. Using a loss-of-function strategy and bioinformatics analysis, we found that MMP-3 expression was regulated by PDE10A in calcifying VSMCs. Our further mechanistic studies showed that PDE10A could regulate vascular calcification by controlling p38 MAPK signaling and MMP-3 activity through cGMP/PKG signaling. These findings suggest that PDE10A plays a critical role in the development of medial artery calcification, and that targeting it may provide a novel therapeutic strategy to reduce medial calcification in patients with PAD and CKD with the ultimate goal of preventing major limb amputation.

Objective: Medial artery calcification is associated with increased cardiovascular morbidity and mortality. It occurs with diabetes and chronic kidney disease. We have previously demonstrated that the matrix metallopeptidase 3 (MMP-3) is strongly induced in arterial calcification. We also showed that MMP-3 inhibition decreases calcium accumulation in vascular SMCs and that MMP-3 deficient mice develop less medial calcification than wildtype controls. In this series of continuing experiments, we evaluate the effects of MMP-3 on SMCs phenotypic transformation in vitro and in vivo. Methods and Results: Confluent rat aortic smooth muscle cells (RASMCs) cultured in calcification medium containing elevated calcium and phosphate levels for 7 days showed increased MMP-3 activity, decreased expression of the SMC markers SM-actin and SM-MHC, and increased expression of the bone markers alkaline phosphatase (ALP) and osterix (Osx). Cells were next exposed a selective MMP-3 inhibitor 2(EMD Millipore). In the presence of inhibitor, MMP-3 activity (MMP-3 activity assay kit, Abcam) was significantly decreased. Additionally, SMC osteogenic transformation was prevented as demonstrated by maintenance of SM22α and SM-MHC expression with reduction of ALP and Osx expression. In confirmatory experiments, MMP-3 reduction with siRNA inhibited the calcification of SMC that were exposed to calcification medium. We next evaluated phenotypic marker expression in MMP-3 knockout and wild-type mice injected with vitamin D 3 , a model of medial artery calcification. At 7 days after injection, expression of the SMC marker gene SM-MHC was significantly greater in MMP-3 KO mice than controls, while bone cell marker genes (Runx2, ALP, Osx) were decreased as measured by qPCR. Deletion of MMP-3 thus inhibited the osteogenic transformation of medial SMCs in vitamin D 3 -treated mice suggesting that it may control calcification via local effects within the arterial wall. Conclusion: Together these findings suggest that MMP-3 promotes medial artery calcification through local effects on the phenotypic state of vascular SMCs, and further, that it may serve as a therapeutic target to reduce calcification and improve outcomes in our PAD patient population.

Objective: Medial artery calcification is associated with increased cardiovascular morbidity and mortality in patients with peripheral artery disease (PAD). It is highly prevalent in diabetes and chronic kidney disease, and it is an important risk factor for cardiovascular events. Accumulating evidences suggest that matrix degrading enzymes are prominently involved in this process. Clinical studies have shown that the matrix metallopeptidase3 (MMP-3, stromelysin-1) is correlated with prevalent arterial calcification in high risk human populations. In this study, we sought to determine whether reducing MMP-3 activity could decrease arterial calcification in smooth muscle cells, organ culture, and in vivo models. Methods and Results: Confluent human aortic smooth muscle cells (HASMCs) were cultured in calcification medium containing high phosphate (Pi) for 7 days in the absence or presence of an MMP-3 specific inhibitor. We found that adding MMP-3 inhibitor dose-dependently reduced Pi-induced calcium deposition as demonstrated by the O-cresolphthalein complexone assay. To further determine the importance of MMP-3 in arterial calcification, we utilized ex vivo aortic rings. In aortic rings from MMP-3 knockout mice, Pi-induced medial calcification was significantly reduced compared with wild-type mice. We next evaluated the effects of MMP-3 deletion in vivo . 12-week-old MMP-3 knockout and wild type mice were injected with 8mg/kg cholecalciferol and arteries were collected after 8 days, Calcium levels were significantly lower in MMP-3 knockout mice. Deletion of MMP-3 also reduced serum calcium levels, but not changed serum phosphate level. In concurrent studies, we showed by immunohistochemical staining that MMP-3 was highly expressed in calcified lesion from human tibial arteries. Conclusion: Together these findings suggest that MMP-3 promotes medial artery calcification, and that it may serve as a potential therapeutic target aimed at improving the poor outcomes of our patients with PAD.

Rationale: Abdominal aortic aneurysms (AAA) are characterized by aortic enlargement and underlying weakness of the vessel wall. Experimental and clinical evidence suggests that vascular inflammation is a central trigger of AAA formation. Phosphodiesterases (PDEs), known regulators of cyclic nucleotide signaling, play a critical role in vascular inflammation. Objective: In this study, we sought to determine the role and function of PDE10A in vascular inflammation and AAA formation. Methods and Results: Extensive evidence suggests that angiotensin II (Ang II) signaling plays an important causative role in AAA formation. Therefore, Real-time PCR array for all 22 known PDE genes was performed in control and Ang II-treated VSMCs. We observed that PDE10A elicited the highest levels of induction by Ang II among all PDEs. Moreover, we found that PDE10A was dramatically upreguated in the Ang II-infused AAA mouse model and in human AAA specimens. PDE10A was primarily expressed in medial VSMCs and infiltrating macrophages in AAA. More importantly, deficiency of PDE10A or PDE10A inhibition significantly attenuated AAA formation in vivo. In cultured VSMCs, knockdown of PDE10A with specific siRNA and inhibition of PDE10A by papaverine markedly suppressed Ang II-induced vascular cell adhesion molecule 1 (VCAM-1), monocyte chemoattractant protein-1 (MCP-1) and MMP2 expression. Deficiency of PDE10A also blocked lipopolysaccharide (LPS)-induced TNF-α, MCP-1, and MMP9 expression in peritoneal macrophages isolated from PDE10A knockout mice. Further mechanistic studies revealed that histone deacetylase 5 (HDAC5) plays an important role in PDE10A-regulated vascular inflammation via cAMP-dependent protein kinase (PKA). Conclusions: These findings demonstrate that PDE10A is an important regulator of vascular inflammation and AAA development. They further provide evidence for PDE10A as a potential therapeutic target for aortic aneurysms and other vascular diseases.

Neointimal hyperplasia is associated with the development of diverse vascular diseases such as atherosclerosis, vein bypass graft disease and restenosis after percutaneous coronary interventions. Vascular injury-mediated neointimal hyperplasia involves the phenotypic modulation, migration and proliferation of vascular smooth muscle cells (VSMCs). Cyclic nucleotide is vital in regulating VSMC migration and proliferation, which are controlled by cyclic nucleotide phosphodiesterase (PDE) isozymes. To identify the regulation and function of PDEs in VSMC pathogenesis of vascular diseases, we performed systematic screening for all 22 known PDE genes in normal contractile VSMCs and proliferating synthetic VSMCs. Interestingly, we observed that expression of PDE1C was very low in normal contractile VSMCs but drastically elevated in synthetic VSMCs in vitro as well as in vivo in various mouse models of vascular injuries, including carotid artery ligation, femoral artery wire injury and vein bypass graft. Consistently, PDE1C is also highly induced in VSMCs in neointimal lesion of human coronary artery. More importantly, injury-induced neointimal formation was significantly attenuated in pde1c-deficient mice or by perivascular administration of PDE1 inhibitor. Furthermore, PDE1 inhibition also reduced the spontaneous vascular remodeling of human saphenous vein explant in an ex vivo culture model. Mechanistic studies revealed that PDE1C plays a critical role in regulating the stability of PDGF receptor beta (PDGFR-β). We found that PDE1C knockdown or inhibition markedly decreased the levels of PDGFR-β protein but not mRNA, which was blocked by endosome and lysosome inhibitors. Furthermore, PDE1C inhibition is capable of promoting PDGFR-β internalization and attenuating PDGFR-β membrane accumulation. Finally, we found that PDE1C co-localized with PDGFR-β on cell membrane, which was able to regulate PDGFR-β protein degradation through modulating a transmembrane adenylyl cyclase-cAMP-PKA signaling. Taken together, our present data demonstrated that PDE1C plays a critical role in regulating VSMC proliferation and neointimal hyperplasia, in part, through promoting endosome/lysosome dependent PDGFR-β protein degradation.