Apoptosis of human endothelial cells after growth factor deprivation is associated with rapid and dramatic up-regulation of cyclin A-associated cyclin-dependent kinase 2(cdk2) activity. In apoptotic cells, the C termini of the cdk inhibitors p21Cip1/Waf1 and p27Kip1 are truncated by specific cleavage. The enzyme involved in this cleavage is CPP32 and/or a CPP32-like caspase. After cleavage, p21Cip1/Waf1 loses its nuclear localization sequence and exits the nucleus. Cleavage of p21Cip1/Waf1 and p27Kip1 results in a substantial reduction in their association with nuclear cyclin-cdk2 complexes, leading to a dramatic induction of cdk2 activity. Dominant-negative cdk2, as well as a mutant of p21Cip1/Waf1 resistant to caspase cleavage, partially suppress apoptosis. These data suggest that cdk2 activation, through caspase-mediated cleavage of cdk inhibitors, may be instrumental in the execution of apoptosis following caspase activation.

Arterial smooth muscle cells (SMCs) are arrested in the G1 phase of the cell cycle on polymerized type I collagen fibrils, while monomer collagen supports SMC proliferation. Cyclin E-associated kinase and cyclin-dependent kinase 2 (cdk2) phosphorylation are inhibited on polymerized collagen, and levels of the cdk2 inhibitors p27Kip1 and p21Cip1/Waf1 are increased compared with SMCs on monomer collagen. p27Kip1 associates with the cyclin E-cdk2-p21Cip1/Waf1 complex in SMCs on polymerized collagen. Monovalent blocking antibodies to alpha2 integrins, integrins that mediate adhesion to both forms of collagen, mimic these effects on monomer collagen. Furthermore, polymerized collagen rapidly suppresses p70 S6 kinase, a possible regulator of p27Kip1. Thus, fibrillar collagen specifically regulates early integrin signaling that may lead to up-regulation of cdk2 inhibitors and inhibition of SMC proliferation.

These results indicate that use of oral alfacalcidol was associated with reduced risk for cardiovascular death in this cohort of ESRD patients. The result of this observational study warrants further randomized controlled trials with 1alpha-hydroxy vitamin D3 to confirm the possibility that such medication improves survival of ESRD patients.

Abstract. Insulin resistance is closely associated with atherosclerosis and cardiovascular mortality in the general population. Patients with end-stage renal disease (ESRD) are known to have insulin resistance, advanced atherosclerosis, and a high cardiovascular mortality rate. We evaluated whether insulin resistance is a predictor of cardiovascular death in a cohort of ESRD. A prospective observational cohort study was performed in 183 nondiabetic patients with ESRD treated with maintenance hemodialysis. Insulin resistance was evaluated by the homeostasis model assessment method (HOMA-IR) using fasting glucose and insulin levels at baseline, and the cohort was followed for a mean period of 67 mo. Forty-nine deaths were recorded, including 22 cardiovascular deaths. Cumulative incidence of cardiovascular death by Kaplan-Meier estimation was significantly different between subjects in the top tertile of HOMA-IR (1.40 to 4.59) and those in the lower tertiles of HOMA-IR (0.28 to 1.39), and the hazard ratio (HR) was 2.60 (95% confidence interval [CI], 1.12 to 6.01; P ϭ 0.026) in the univariate Cox proportional hazards model. In multivariate Cox models, the positive association between HOMA-IR and cardiovascular mortality remained significant (HR, 4.60; 95% CI, 1.83 to 11.55; P ϭ 0.001) and independent of age, C-reactive protein, and presence of preexisting vascular complications. Further analyses showed that the effect of HOMA-IR on cardiovascular mortality was independent of body mass index, hypertension, and dyslipidemia. In contrast, HOMA-IR did not show such a significant association with noncardiovascular mortality. These results indicate that insulin resistance is an independent predictor of cardiovascular mortality in ESRD.

Calcification is a common feature of advanced atherosclerotic lesions and is being reemphasized as a clinically significant element of vascular disease. However, the scarcity of in vitro models of vascular calcification preclude studying its molecular and cellular mechanism. In the present study, we describe an in vitro calcification in which diffuse calcification can be induced by culturing bovine vascular smooth muscle cells (BVSMC) in the presence of beta-glycerophosphate, ascorbic acid, and insulin in a manner analogous to in vitro mineralization by osteoblasts. Calcification was confirmed by von Kossa staining and 45Ca accumulation. Factor analysis revealed that beta-glycerophosphate is the most important factor for this calcification process, suggesting that alkaline phosphatase (ALP) may be involved. As predicted, high levels of ALP expression were detected by ALP assay and Northern blot analysis. Functional significance of ALP was confirmed by demonstrating that levamisole, a specific inhibitor of ALP, inhibited BVSMC calcification in a dose-dependent manner. Bisphosphonates such as etidronate and pamidronate potently inhibited BVSMC calcification, suggesting that hydroxyapatite formation may be involved. Importantly, expression of osteopontin mRNA was dramatically increased in calcified BVSMC compared with uncalcified control cells. These data suggest that beta-glycerophosphate can induce diffuse calcification by an ALP-dependent mechanism and that this in vitro calcification system is useful for analyzing the molecular and cellular mechanisms of vascular calcification.