Background— Given the central importance of nitric oxide (NO) in the development and clinical course of cardiovascular diseases, we sought to determine whether the powerful predictive value of C-reactive protein (CRP) might be explained through an effect on NO production. Methods and Results— Endothelial cells (ECs) were incubated with recombinant CRP (0 to 100 μg/mL, 24 hours), and NO and cyclic guanosine monophosphate (cGMP) production was assessed. The effects of CRP on endothelial NO synthase (eNOS) protein, mRNA expression, and mRNA stability were also examined. In a separate study, the effects of CRP (25 μg/mL) on EC cell survival, apoptosis, and in vitro angiogenesis were evaluated. Incubation of ECs with CRP resulted in a significant inhibition of basal and stimulated NO release, with concomitant reductions in cGMP production. CRP caused a marked downregulation of eNOS mRNA and protein expression. Actinomycin D studies suggested that eNOS downregulation was related to decreased mRNA stability. In conjunction with a decrease in NO production, CRP inhibited both basal and vascular endothelial growth factor–stimulated angiogenesis as assessed by EC migration and capillary-like tube formation. CRP did not induce EC survival but did, however, promote apoptosis in a NO-dependent fashion. Conclusions— CRP, at concentrations known to predict adverse vascular events, directly quenches the production of the NO, in part, through posttranscriptional effect on eNOS mRNA stability. Diminished NO bioactivity, in turn, inhibits angiogenesis, an important compensatory mechanism in chronic ischemia. Through decreasing NO synthesis, CRP may facilitate the development of diverse cardiovascular diseases. Risk reduction strategies designed to lower plasma CRP may be effective by improving NO bioavailability.

Ou are contacted by a concerned 34-year-old airline pilot with a leaky bicuspid aortic valve recently diagnosed by an echocardiogram that had been requested by his new employer as part of a routine medical assessment. He claims that he is perfectly healthy but is at risk of losing his job over his condition. He is concerned that his disease is hereditary and that his children may also be at risk. The patient, his attorney, and his insurance company have requested a statement from you as to the cause, possible complications, and treatment options associated with a con- genital malformation of the aortic valve. The bicuspid aortic valve (BAV) is the most common congenital cardiac malformation, occurring in 1% to 2% of the population. The majority of BAV patients develop com- plications requiring treatment. Physicians are often chal- lenged when asked to provide evidence-based advice about BAV disease because the pathogenesis and pathophysiology of this disease are not well understood.

Background— Adipocyte-derived hormones may represent a mechanism linking insulin resistance to cardiovascular disease. In the present study, we evaluated the direct effects of resistin, a novel adipocyte-derived hormone, on endothelial activation. Methods and Results— Endothelial cells (ECs) were incubated with human recombinant resistin (10 to 100 ng/ML, 24 hours), and endothelin-1 (ET-1) release, ET-1 mRNA expression, and nitric oxide (NO) production were assessed. Transient transfection assays were used to evaluate the effects of resistin on transcription of human ET-1 gene promoter. Furthermore, the effects of resistin on AP-1–mutated ET-1 promoter were evaluated. The effects of resistin on expression of vascular cell adhesion molecule (VCAM-1) and monocyte chemoattractant chemokine (MCP-1) were studied in addition to CD40 receptor, CD40 ligand–induced MCP-1 expression, and tumor necrosis factor receptor–associated factor-3 (TRAF3), an inhibitor of CD40 signaling. Incubation of ECs with resistin resulted in an increase in ET-1 release and ET-1 mRNA expression, with no change in NO production. Whereas treatment with resistin resulted in an increase in ET-1 promoter activity, the AP-1–mutated promoter was inactive after resistin stimulation. Additionally, resistin-treated cells showed increased expression of VCAM-1 and MCP-1, with concomitant reductions in TRAF-3 expression. Resistin did not alter CD40 receptor expression; however, increased CD40 ligand induced MCP-1 production. Conclusions— The novel adipokine resistin exerts direct effects to promote EC activation by promoting ET-1 release, in part by inducing ET-1 promoter activity via the AP-1 site. Furthermore, resistin upregulates adhesion molecules and chemokines and downregulates TRAF-3, an inhibitor of CD40 ligand signaling. In this fashion, resistin may be mechanistically linked to cardiovascular disease in the metabolic syndrome.

Background — C-reactive protein (CRP) has been suggested to actively participate in the development of atherosclerosis. In the present study, we examined the role of the potent endothelium-derived vasoactive factor endothelin-1 (ET-1) and the inflammatory cytokine interleukin-6 (IL-6) as mediators of CRP-induced proatherogenic processes. Methods and Results — Saphenous vein endothelial cells (HSVECs) were incubated with human recombinant CRP (25 μg/mL, 24 hours) and the expression of vascular cell adhesion molecule (VCAM-1), intracellular adhesion molecule (ICAM-1), and monocyte chemoattractant chemokine-1 was determined. The effects of CRP on LDL uptake were assessed in macrophages using immunofluorescent labeling of CD32 and CD14. In each study, the effect of endothelin antagonism (bosentan) and IL-6 inhibition (monoclonal anti-IL-6 antibodies) was examined. The effects of CRP on the secretion of ET-1 and IL-6 from HSVECs were also evaluated. Incubation of HSVECs with recombinant human CRP resulted in a marked increase in ICAM-1 and VCAM-1 expression ( P <0.001). Likewise, CRP caused a significant increase in monocyte chemoattractant chemokine-1 production, a key mediator of leukocyte transmigration ( P <0.001). CRP caused a marked and sustained increase in native LDL uptake by macrophages ( P <0.05). These proatherosclerotic effects of CRP were mediated, in part, via increased secretion of ET-1 and IL-6 ( P <0.01) and were attenuated by both bosentan and IL-6 antagonism ( P <0.01). Conclusions — CRP actively promotes a proatherosclerotic and proinflammatory phenotype. These effects are mediated, in part, via the production of ET-1 and IL-6 and are attenuated by mixed ET _A/B receptor antagonism and IL-6 inhibition. Bosentan may be useful in decreasing CRP-mediated vascular disease.

Background— Myocardial ischemia provides a potent stimulus to angiogenesis, and the mobilization and differentiation of endothelial progenitor cells (EPCs) has been shown to be important in this process. An elevated level of C-reactive protein (CRP) has emerged as one of the most powerful predictors of cardiovascular disease. However, the impact of CRP on EPC biology is unknown. Methods and Results— EPCs were isolated from the peripheral venous blood of healthy male volunteers. Cells were cultured in endothelial cell basal medium-2 in the absence and presence of CRP (5 to 20 μg/mL), rosiglitazone (1 μmol/L), and/or vascular endothelial growth factor. EPC differentiation, survival, and function were assayed. CRP at concentrations ≥15 μg/mL significantly reduced EPC cell number, inhibited the expression of the endothelial cell–specific markers Tie-2, EC-lectin, and VE-cadherin, significantly increased EPC apoptosis, and impaired EPC-induced angiogenesis. EPC-induced angiogenesis was dependent on the presence of nitric oxide, and CRP treatment caused a decrease in endothelial nitric oxide synthase mRNA expression by EPCs. However, all of these detrimental CRP-mediated effects on EPCs were attenuated by pretreatment with rosiglitazone, a peroxisome proliferator–activated receptor-γ (PPARγ) agonist. Conclusions— Human recombinant CRP, at concentrations known to predict adverse vascular outcomes, directly inhibits EPC differentiation, survival, and function, key components of angiogenesis and the response to chronic ischemia. This occurs in part via an effect of CRP to reduce EPC eNOS expression. The PPARγ agonist rosiglitazone inhibits the negative effects of CRP on EPC biology. The ability of CRP to inhibit EPC differentiation and survival may represent an important mechanism that further links inflammation to cardiovascular disease.

Clinical trials of bone marrow stem/progenitor cell therapy after myocardial infarction (MI) have shown promising results, but the mechanism of benefit is unclear. We examined the nature of endogenous myocardial repair that is dependent on the function of the c-kit receptor, which is expressed on bone marrow stem/progenitor cells and on recently identified cardiac stem cells. MI increased the number of c-kit⁺ cells in the heart. These cells were traced back to a bone marrow origin, using genetic tagging in bone marrow chimeric mice. The recruited c-kit⁺ cells established a proangiogenic milieu in the infarct border zone by increasing VEGF and by reversing the cardiac ratio of angiopoietin-1 to angiopoietin-2. These oscillations potentiated endothelial mitogenesis and were associated with the establishment of an extensive myofibroblast-rich repair tissue. Mutations in the c-kit receptor interfered with the mobilization of the cells to the heart, prevented angiogenesis, diminished myofibroblast-rich repair tissue formation, and led to precipitous cardiac failure and death. Replacement of the mutant bone marrow with wild-type cells rescued the cardiomyopathic phenotype. We conclude that, consistent with their documented role in tumorigenesis, bone marrow c-kit⁺ cells act as key regulators of the angiogenic switch in infarcted myocardium, thereby driving efficient cardiac repair.