Mitesh Badiwala

A Self-Fulfilling Prophecy C-Reactive Protein Attenuates Nitric Oxide Production and Inhibits Angiogenesis

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 &mgr;g/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 &mgr;g/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.

Endothelin Antagonism and Interleukin-6 Inhibition Attenuate the Proatherogenic Effects of C-Reactive Protein

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 &mgr;g/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 ETA/B receptor antagonism and IL-6 inhibition. Bosentan may be useful in decreasing CRP-mediated vascular disease.

Hyperglycemia potentiates the proatherogenic effects of C-reactive protein: reversal with rosiglitazone

Accumulating evidence suggests that C-reactive protein (CRP), at concentrations known to predict diverse vascular insults, directly promotes endothelial cell activation, uncovering a proatherosclerotic and proinflammatory phenotype. In the present study, we hypothesized that (a). hyperglycemia would serve to exaggerate the proatherogenic effects of CRP and (b). the PPARgamma agonist, rosiglitazone would attenuate this effect. Human saphenous vein endothelial cells were studied under the following conditions (n= 10 per group): control, human recombinant CRP (25 microg/ml, 24 h), hyperglycemia (25 mM, 24 h) and hyperglycemia + CRP. In each case, the effects of co-incubation with rosiglitazone (1 microM) were evaluated. Nitric oxide and endothelin-1 release from endothelial cells was quantified, in addition to the expression of adhesion molecules and monocyte chemoattractant chemokine (MCP-1). Incubation of endothelial cells with CRP increased endothelin-1 production, and upregulated adhesion molecule and MCP-1 expression. These proatherogenic effects of CRP were potentiated in the presence of hyperglycemia. CRP also decreased endothelial nitric oxide release, and this effect remained unchanged by hyperglycemia. Importantly, the PPARgamma agonist, rosiglitazone, attenuated the proatherogenic effects of CRP under both basal and hyperglycemic conditions. The direct proatherogenic actions of CRP are exaggerated in the hyperglycemic milieu, and attenuated by rosiglitazone. Elevated CRP levels in patients with diabetes may serve to uncover a severe proatherogenic phenotype.

Predictors of low cardiac output syndrome after isolated mitral valve surgery

BACKGROUND Low cardiac output syndrome is defined as the need for a postoperative intra-aortic balloon pump or inotropic support for longer than 30 minutes in the intensive care unit. Mitral valve surgery is increasingly being performed in high-risk patients who might require mechanical circulatory support for low cardiac output syndrome. Therefore the aim of this study was to identify the preoperative predictors of low cardiac output syndrome after mitral valve surgery. METHODS We conducted a retrospective review of data prospectively entered into an institutional database. Between 1990 and February 2008, 3039 patients underwent isolated mitral valve surgery with or without coronary bypass surgery. The independent predictors of low cardiac output syndrome and operative mortality were determined by means of stepwise logistic regression analysis. RESULTS The overall prevalence of low cardiac output syndrome was 7%. The independent predictors of low cardiac output syndrome were urgency of the operation (odds ratio, 2.9), earlier year of operation (odds ratio, 2.4), left ventricular ejection fraction of less than 40% (odds ratio, 2.1), New York Heart Association class IV (odds ratio, 2), body surface area of 1.7 m(2) or less (odds ratio, 1.6), ischemic mitral valve pathology (odds ratio, 1.6), and cardiopulmonary bypass time (odds ratio, 1.02). The operative mortality was higher in patients with low cardiac output syndrome (30% vs 1.3%, P < .001). Overall operative mortality was 3.4%. The independent predictors of mortality were urgency of the operation (odds ratio, 7.1), renal failure (odds ratio, 4.3), nonuse of polytetrafluoroethylene sutures (Gore-Tex; W. L. Gore & Associates, Inc, Austin, Tex; odds ratio, 2.1), any reoperative surgical intervention (odds ratio, 1.8), increasing age (odds ratio, 1.03), and cardiopulmonary bypass time (odds ratio, 1.02). CONCLUSIONS Low cardiac output syndrome is associated with significantly increased morbidity and mortality. Novel strategies to preserve renal function, optimization of pre-existing heart failure symptoms, and use of artificial polytetrafluoroethylene sutures might reduce the incidence of low cardiac output syndrome and lead to improved results after mitral valve surgery.

Tetrahydrobiopterin attenuates homocysteine induced endothelial dysfunction.

Homocysteine is an independent risk factor for atherosclerotic vascular disease. It impairs endothelial function via increasing superoxide production and quenching nitric oxide (NO) release. Tetrahydrobiopterin (BH4) is a critical cofactor that couples nitric oxide synthase and facilitates the production of nitric oxide (vs. superoxide anions). In the first study, the effects of hyperhomocysteinemia (0.1 mM, 3 h) on endothelium-dependent vasorelaxation to ACh and A23187 were examined in isolated segments of rat aortae in the presence or absence of BH4 (0.1 mM). In the second study, the effects of hyperhomocysteinemia (24 h) on nitric oxide production and superoxide release (using lucigenin chemiluminescence) were studied in human umbilical vein endothelial cells in the absence or presence of BH4 (10 μM). Homocysteine incubation impaired receptor-dependent and -independent endothelial function to ACh and A23187. This effect was attenuated by BH4. Furthermore, homocysteine exposure increased superoxide production and impaired agonist-stimulated nitric oxide release. These effects were attenuated by BH4 (p < 0.05). Hyperhomocysteinemia impairs endothelial function, in part due to a diminished bioavailability of BH4 with resultant uncoupling of nitric oxide synthase. BH4 may represent an important target for strategies aimed at improving endothelial dysfunction secondary to hyperhomocysteinemia.

Progesterone Induces Human Leukocyte Antigen-G Expression in Vascular Endothelial and Smooth Muscle Cells

Background— Human leukocyte antigen-G (HLA-G) expression in heart transplant patients has been negatively associated with acute cellular rejection and cardiac allograft vasculopathy. We assessed HLA-G expression in vascular human endothelial and smooth muscle cell cultures to determine if future therapeutic agents can be targeted toward inducing HLA-G expression to protect against allograft rejection and vasculopathy. Methods and Results— Human coronary artery endothelial, aortic endothelial, and coronary artery smooth muscle cell cultures were exposed to cytokines (interferon-γ or interleukin-10), hypoxia/reoxygenation stress, immunosuppressive agents (cyclosporine, sirolimus, or tacrolimus), or progesterone. HLA-G was not expressed by untreated, normoxic cells. Furthermore, maximal doses of interferon-γ, interleukin-10, cyclosporine, sirolimus, or tacrolimus, as well as exposure to hypoxia/reoxygenation, failed to induce HLA-G expression. HLA-G, which has previously not been detected in adult vascular endothelial and smooth muscle cells, was detected by enzyme-linked immunosorbent assay and flow cytometry in human coronary artery endothelial, human coronary aortic endothelial, and human coronary artery smooth muscle cultures after incubation with progesterone in a dose-dependent manner (P<0.001) with no change in cellular proliferation ability or viability. This effect was partially blocked in the presence of mifepristone, a progesterone receptor antagonist (human coronary artery endothelial: 48.8±15.6%; human coronary aortic endothelial: 59.5±9.5%; human coronary artery smooth muscle: 59.8±9.8% of control; P<0.05). Progesterone-induced HLA-G expression was not protective against hypoxia/reoxygenation injury. Conclusions— HLA-G is not expressed at baseline in vascular endothelial and smooth muscle cells but can be induced by exposure to progesterone. Although tightly regulated, induction of HLA-G expression in these cells may represent a promising and novel therapeutic strategy to protect against rejection and cardiac allograft vasculopathy after heart transplantation.

Epidermal Growth Factor-Like Domain 7 Suppresses Intercellular Adhesion Molecule 1 Expression in Response to Hypoxia/Reoxygenation Injury in Human Coronary Artery Endothelial Cells

Background— Epidermal growth factor-like domain 7 (Egfl7) is a chemoattractant for endothelial cells, and its expression is restricted to endothelial cells. Hypoxia/reoxygenation (H/R) induced endothelial injury that occurs during transplantation contributes to the subsequent development of allograft vasculopathy. We investigated the effect of Egfl7 on endothelial cell intercellular adhesion molecule 1 expression in response to H/R injury. Methods and Results— Human coronary artery endothelial cells were submitted to hypoxia (0.1% O2) followed by normoxia (21% O2) in the presence or absence of Egfl7 (100 ng/mL). Hypoxia alone increased the expression of Egfl7×140±8% of control at 3 hours (n=6; P<0.05) and 385±50% of control at 6 hours (n=6; P<0.001). Incubation with Egfl7 during the reoxygenation period prevented intercellular adhesion molecule 1 upregulation (mean fluorescence intensity: 5.37±0.92 versus 3.81±0.21; P<0.05; n=4 per group). Nuclear factor-&kgr;B nuclear localization on H/R injury was blocked by Egfl7 administration (cytosolic/nuclear ratio of 0.93±0.01 versus 1.44±0.24; P<0.05; n=4 per group). Inhibitor of nuclear factor-&kgr;B protein level was significantly reduced on H/R injury (26±4.6% of control expression; P<0.05; n=4 per group); however, concurrent incubation with Egfl7 attenuated this reduction (46±6.2% of control expression; P<0.05 when compared with H/R injury alone; n=4 per group). Conclusions— Our study reveals the novel observation that hypoxia upregulates human coronary artery endothelial cells expression of Egfl7 and that Egfl7 inhibits expression of intercellular adhesion molecule 1 subsequent to H/R injury. Mechanistically, Egfl7 prevented nuclear factor-&kgr;B nuclear localization and augmented inhibitor of nuclear factor-&kgr;B protein levels, suggesting that it inhibits nuclear factor-&kgr;B activation, a key step in the inflammatory activation of endothelial cells. Egfl7 may be protective against H/R injury incurred during transplantation and may modulate the events that lead to the development of graft vasculopathy.

Epidermal Growth Factor–Like Domain 7 Is a Novel Inhibitor of Neutrophil Adhesion to Coronary Artery Endothelial Cells Injured by Calcineurin Inhibition

Background— We investigated the effect of epidermal growth factor–like domain 7 (Egfl7) on nuclear factor-&kgr;B activation, intercellular adhesion molecule-1 expression, and neutrophil adhesion to human coronary artery endothelial cells after calcineurin-inhibition–induced injury. Methods and Results— Human coronary endothelial cells were incubated with cyclosporine (CyA) 10 &mgr;g/mL with or without Egfl7 (100 ng/mL) or the Notch receptor activator Jagged1 (200 ng/mL) for 6 to 48 hours. CyA upregulated nuclear factor-&kgr;B (p65) activity (128±2% of control, P<0.001) in nuclear extracts, as determined with a DNA-binding activity ELISA. This activity was inhibited by Egfl7 (86±3% of control; P<0.001 versus CyA alone). Jagged1 blocked Egfl7-induced nuclear factor-&kgr;B inhibition (105±4% of control; P<0.05 versus CyA plus Egfl7). CyA upregulated cell-surface intercellular adhesion molecule-1 expression (215±13% of control; P<0.001), as determined by flow cytometry. This expression was suppressed by Egfl7 (148±5%; P<0.001 versus CyA alone). Jagged1 attenuated the intercellular adhesion molecule-1–suppressive effect of Egfl7 when administered with CyA (193±3% versus 148±5%; P<0.01). CyA increased neutrophil adhesion to human coronary endothelial cells (control 20±5%, CyA 37±3%; P<0.001 versus control) in a nonstatic neutrophil adhesion assay. This increase was attenuated by Egfl7 (22±6%; P<0.001 versus CyA alone). Jagged 1 attenuated the effect of Egfl7 on neutrophil adhesion (31±3%; P<0.001 versus Egfl7 plus CyA). Conclusions— Our study reveals that Egfl7 is a potent inhibitor of neutrophil adhesion to human coronary endothelial cells subsequent to calcineurin-inhibition–induced injury. Mechanistically, Egfl7 blocked nuclear factor-&kgr;B pathway activation and intercellular adhesion molecule-1 expression, which suggests that it may have significant antiinflammatory properties. Because Jagged1 blocked the effect of Egfl7, Notch receptor antagonism may contribute to the mechanism of action of Egfl7.

Left ventricular device as destination therapy: are we there yet?

Purpose of review Mechanical circulatory assistance is evolving from bridge-to-transplant to destination therapy for end-stage heart failure patients. Recent studies document the evolution in outcomes following destination therapy achieved subsequent to the landmark Randomized Evaluation of Mechanical Assistance for the Treatment of Congestive Heart Failure trial, as well as the outcomes of second-generation left ventricular assist devices (LVADs). Recent findings Post-Randomized Evaluation of Mechanical Assistance for the Treatment of Congestive Heart Failure destination therapy patients receiving first-generation pulsatile flow devices have benefited from some improvement in 2-year outcomes and some reduction in the incidence of adverse events including device failure and sepsis. Despite these improvements, survival at 2 years remains poor and adverse events continue to portend significant morbidity and mortality. Second-generation continuous axial flow devices have demonstrated superior device durability and reliability as well as significant reductions in the incidence of sepsis and device-related infections in bridge-to-transplant patients. Summary Second-generation axial flow devices have significantly improved the adverse-event profile of LVAD therapy and are anticipated to offer substantial device longevity. Destination therapy is clearly an option for some patients with end-stage heart failure. As devices and experience evolve, outcomes will improve, which begs the question of whether these devices will have a role in the treatment of patients not as ill as those in the Randomized Evaluation of Mechanical Assistance for the Treatment of Congestive Heart Failure trial.

Mechanical circulatory support with the ABIOMED BVS 5000: the Toronto General Hospital experience.

BACKGROUND Acute hemodynamic collapse resulting in cardiogenic shock and impending end-organ failure is usually associated with certain death. The introduction of short-term mechanical circulatory support (MCS) devices offers potential therapy to these critically ill patients. The BVS 5000 device (ABIOMED Inc, USA) is widely used in the United States, but rarely in Canada, where device reimbursement remains a barrier. OBJECTIVE To present the Toronto General Hospitals (Toronto, Ontario) initial five-year experience with this device to highlight the indications for use, common complications and overall success rates. METHODS AND RESULTS The institutional MCS database from 2001 to 2006 was reviewed, and 18 patients who received 30 devices in a variety of configurations were identified. The most common support configuration consisted of biventricular support (n=12), followed by isolated left ventricular support (n=4) and isolated right ventricular support in two recipients of an implantable long-term left ventricular assist device. Overall survival to device explant or transplant was 55% (n=10), of which five (50%) were successfully discharged from the hospital. The overall survival from device implant to hospital discharge was 28% (five of 18). The most common cause of death was multisystem organ failure. CONCLUSIONS MCS with the ABIOMED BVS 5000 can successfully resuscitate critically ill patients; however, earlier institution of this device would avoid irreversible end-organ injury, and lead to higher rates of device explant and hospital discharge. Short-term MCS devices should be available in all cardiac surgical centres in Canada to permit stabilization and evaluation of the acutely ill cardiac patient and subsequent management in a heart transplant facility.