Circulating endothelial cells as predictor of long-term mortality and adverse cardiovascular outcomes in hemodialysis patients.

Circulating endothelial cells (CEC) are thought to be markers of endothelial injury. We hypothesized that the numbers of CEC may provide a novel means for predicting long-term survival and cardiovascular events in hemodialysis patients. 54 hemodialysis patients underwent enumeration of their CEC number. We retrospectively analyzed their survival and incidence of adverse cardiovascular events. 22 deaths (41%) were noted over the median follow up period of 3.56 years (IQR 1.43-12) and 6 were attributed to cardiovascular deaths (11%) of which 1 (4%) was in the low CEC (CEC<20 cells/ml) and 5 (19%) in the high CEC (CEC≥20 cells/ml) group. High CEC was associated with worse cardiovascular survival (p = 0.05) and adverse cardiac events (p = 0.01). In multivariate analysis, CEC >20 cells/ml was associated with a 4-fold increased risk of adverse cardiac events (OR, 4.16 [95% CI,1.38-12.54],p = 0.01) while all-cause mortality and cardiovascular mortality were not statistically different. In this hemodialysis population, a single measurement of CEC was a strong predictor of long term future adverse cardiovascular events. We propose that CEC may be a novel biomarker for assessing cardiovascular risk in dialysis patients.

Potential therapeutic use of relaxin in accelerating closure of cranial bone defects in mice.

Bone fractures are associated with considerable morbidity and increased mortality. A major limitation to healing is lack of bone blood flow, which is impaired by physical disruption of intraskeletal and/or periosteal vasculature by the fracture. Thus, pharmacological interventions are needed to improve osseous blood flow, thereby accelerating bone fracture closure. Relaxin is secreted by the ovary and circulates in rodents and humans during pregnancy. Because relaxin might benefit bone fracture healing by stimulating angiogenesis, vasculogenesis (and potentially osteogenesis) through mobilization and activation of bone marrow progenitor cells, and by increasing blood flow via vasodilation, we investigated whether relaxin administration would accelerate closure of a calvarial defect in mice. Whether administered systemically by osmotic pump or locally by collagen scaffolds for ~2 week period after lesioning, relaxin did not accelerate bone healing. Despite implementing relaxin doses that reached plasma concentrations spanning the physiological to supraphysiological range, testing the closure of two different sizes of calvarial lesions, allowing for different intervals of time from instigation of cranial lesion to euthanasia, and investigating mice of different ages, we did not observe a significant benefit of relaxin in bone lesion healing. Nor did we observe stimulation of blood vessel formation in the bone lesion by the hormone. An incidental finding was that relaxin appeared to enhance trabecular bone growth in an uninjured control bone (femur). Although the results of this study were not supportive of a therapeutic benefit for relaxin on calvarial defect closure, future investigation is needed employing different animal species and experimental models of bone fracture.

Activation of the ß-common receptor by erythropoietin impairs acetylcholine-mediated vasodilation in mouse mesenteric arterioles.

Clinically, erythropoietin (EPO) is known to increase systemic vascular resistance and arterial blood pressure. However, EPO stimulates the production of the potent vasodilator, nitric oxide (NO), in culture endothelial cells. The mechanism by which EPO causes vasoconstriction despite stimulating NO production may be dependent on its ability to activate two receptor complexes, the homodimeric EPO (EPOR2 ) and the heterodimeric EPOR/ß-common receptor (ßCR). The purpose of this study was to investigate the contribution of each receptor to the vasoactive properties of EPO. First-order, mesenteric arteries were isolated from 16-week-old male C57BL/6 mice, and arterial function was studied in pressure arteriographs. To determine the contribution of each receptor complex, EPO-stimulating peptide (ESP), which binds and activates the heterodimeric EPOR/ßCR complex, and EPO, which activates both receptors, were added to the arteriograph chamber 20 min prior to evaluation of endothelium-dependent (acetylcholine, bradykinin, A23187) and endothelium-independent (sodium nitroprusside) vasodilator responses. Only ACh-induced vasodilation was impaired in arteries pretreated with EPO or ESP. EPO and ESP pretreatment abolished ACh-induced vasodilation by 100% and 60%, respectively. EPO and ESP did not affect endothelium-independent vasodilation by SNP. Additionally, a novel ßCR inhibitory peptide (ßIP), which was computationally developed, prevented the impairment of acetylcholine-induced vasodilation by EPO and ESP, further implicating the EPOR/ßCR complex. Last, pretreatment with either EPO or ESP did not affect vasoconstriction by phenylephrine and KCl. Taken together, these findings suggest that acute activation of the heterodimeric EPOR/ßCR in endothelial cells leads to a selective impairment of ACh-mediated vasodilator response in mouse mesenteric resistance arteries.

Computational design and experimental characterization of a novel ß-common receptor inhibitory peptide.

In short-term animal models of ischemia, erythropoietin (EPO) signaling through the heterodimeric EPO receptor (EPOR)/ß-common receptor (ßCR) is believed to elicit tissue protective effects. However, large, randomized, controlled trials demonstrate that targeting a higher hemoglobin level by administering higher doses of EPO, which are more likely to activate the heterodimeric EPOR/ßCR, is associated with an increase in adverse cardiovascular events. Thus, inhibition of long-term activation of the ßCR may have therapeutic implications. This study aimed to design and evaluate the efficacy of novel computationally designed ßCR inhibitory peptides (ßIP). These novel ßIPs were designed based on a truncated portion of Helix-A from EPO, specifically residues 11-26 (VLERYLLEAKEAEKIT). Seven novel peptides (P1 to P7) were designed. Peptide 7 (P7), VLERYLHEAKHAEKIT, demonstrated the most robust inhibitory activity. We also report here the ability of P7 to inhibit ßCR-induced nitric oxide (NO) production and angiogenesis in human umbilical vein endothelial cells (HUVECs). Specifically, we found that P7 ßIP completely abolished EPO-induced NO production. The inhibitory effect could be overcome with super physiological doses of EPO, suggesting a competitive inhibition. ßCR-induced angiogenesis in HUVEC's was also abolished with treatment of P7 ßIP, but P7 ßIP did not inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis. In addition, we demonstrate that the novel P7 ßIP does not inhibit EPO-induced erythropoiesis with use of peripheral blood mononuclear cells (PBMCs). These results, for the first time, describe a novel, potent ßCR peptide inhibitor that inhibit the actions of the ßCR without affecting erythropoiesis.

Conditional Deletion of Bmal1 Accentuates Microvascular and Macrovascular Injury.

The brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (BMAL)-1 constitutes a major transcriptional regulator of the circadian clock. Here, we explored the impact of conditional deletion of Bmal1 in endothelium and hematopoietic cells in murine models of microvascular and macrovascular injury. We used two models of Bmal1fx/fx;Tek-Cre mice, a retinal ischemia/reperfusion model and a neointimal hyperplasia model of the femoral artery. Eyes were enumerated for acellular capillaries and were stained for oxidative damage markers using nitrotyrosine immunohistochemistry. LSK (lineage-negative, stem cell antigen-1-positive, c-Kit-positive) cells were quantified and proliferation assessed. Hematopoiesis is influenced by innervation to the bone marrow, which we assessed using IHC analysis. The number of acellular capillaries increased threefold, and nitrotyrosine staining increased 1.5-fold, in the retinas of Bmal1fx/fx;Tek-Cre mice. The number of LSK cells from the Bmal1fx/fx;Tek-Cre mice decreased by 1.5-fold and was accompanied by a profound decrease in proliferative potential. Bmal1fx/fx;Tek-Cre mice also exhibited evidence of bone marrow denervation, demonstrating a loss of neurofilament-200 staining. Injured femoral arteries showed a 20% increase in neointimal hyperplasia compared with similarly injured wild-type controls. Our study highlights the importance of the circadian clock in maintaining vascular homeostasis and demonstrates that specific deletion of BMAL1 in endothelial and hematopoietic cells results in phenotypic features similar to those of diabetes.

Effects of Long-Term Type I Interferon on the Arterial Wall and Smooth Muscle Progenitor Cells Differentiation.

OBJECTIVE: Patients with systemic lupus erythematosis are at risk for premature atherosclerosis and half of the patients with systemic lupus erythematosis have elevated type I interferon (IFN-I) levels. We hypothesized that IFN-I would induce premature atherosclerosis by increasing the number of smooth muscle progenitor cells (SMPC) in the bloodstream and promoting atherosclerotic lesions within the vasculature. APPROACH AND RESULTS: SMPC isolated from wild-type and IFN receptor knockout animals were cultured in medium±IFN-I. In vivo, we used electroporation to generate stable IFN-I expression for as long as 4 months. The number of SMPC was determined in mice that expressed IFN-I and in control mice and sections from the bifurcation of the abdominal aorta were analyzed 3 months after electroporation of an IFN-I expression plasmid or a control plasmid. Adding IFN-I to the media increased the number of cultured wild-type SMPC and increased mRNA for SM22, but had no effect on SMPC isolated from IFN receptor knockout mice. Our in vivo results demonstrated a positive relationship between the preatherosclerotic-like lesions and endothelial damage. Although, there were no significant differences in smooth muscle cell density or thickness of the medial layer between groups, the IFN-I-expressing mice had a significant increase in preatherosclerotic-like lesions and immature smooth muscle cells, cells that expressed CD34 and smooth muscle α-actin; but lacked smooth muscle myosin heavy chain. CONCLUSIONS: IFN-I seems to enhance SMPC number in vitro. In vivo IFN-I expression may maintain SMPC in an immature state. These immature smooth muscle cells could give rise to macrophages and eventually foam cells.

Relaxin increases human endothelial progenitor cell NO and migration and vasculogenesis in mice.

The ovarian peptide hormone, relaxin, circulates during pregnancy, contributing to profound maternal vasodilation through endothelial and nitric oxide (NO)-dependent mechanisms. Circulating numbers of bone marrow-derived endothelial cells (BMDECs), which facilitate angiogenesis and contribute to repair of vascular endothelium, increase during pregnancy. Thus, we hypothesized that relaxin enhances BMDEC NO production, circulating numbers, and function. Recombinant human relaxin-2 (rhRLX) stimulated PI3K/Akt B-dependent NO production in human BMDECs within minutes, and activated BMDEC migration that was inhibited by L-N(G)-nitroarginine methyl ester. In BMDECs isolated from relaxin/insulin-like family peptide receptor 2 gene (Rxfp2) knockout and wild-type mice, but not Rxfp1 knockout mice, rhRLX rapidly increased NO production. Similarly, rhRLX increased circulating BMDEC number in Rxfp2 knockout and wild-type mice, but not Rxfp1 knockout mice as assessed by colony formation and flow cytometry. Taken together, these results indicate that relaxin effects BMDEC function through the RXFP1 receptor. Finally, both vascularization and incorporation of GFP-labeled BMDECs were stimulated in rhRLX-impregnated Matrigel pellets implanted in mice. To conclude, relaxin is a novel regulator of BMDECs number and function, which has implications for angiogenesis and vascular remodeling in pregnancy, as well as therapeutic potential in vascular disease.

Induction of nitric oxide by erythropoietin is mediated by the {beta} common receptor and requires interaction with VEGF receptor 2.

Vascular endothelial growth factor (VEGF) and erythropoietin (EPO) have profound effects on the endothelium and endothelial progenitor cells (EPCs), which originate from the bone marrow and differentiate into endothelial cells. Both EPO and VEGF have demonstrated an ability to increase the number and performance properties of EPCs. EPC behavior is highly dependent on nitric oxide (NO), and both VEGF and EPO can stimulate intracellular NO. EPO can bind to the homodimeric EPO receptor (EPO-R) and the heterodimeric receptor, EPO-R and the common beta receptor (betaC-R). Although VEGF has several receptors, VEGF-R2 appears most critical to EPC function. We demonstrate that EPO induction of NO is dependent on the betaC-R and VEGF-R2, that VEGF induction of NO is dependent on the expression of the betaC-R, and that the betaC-R and VEGF-R2 interact. This is the first definitive functional and structural evidence of an interaction between the 2 receptors and has implications for the side effects of EPO.

The role of RAGE in aminoguanidine-induced suppression of venous intimal hyperplasia in diabetic rats.

Intimal hyperplasia is one of the major pathological processes in vein graft failure with diabetes mellitus. In this study, we tested the hypothesis that the suppressive effect of aminoguanidine on intimal hyperplasia is mediated by downregulated expression of advanced glycation end products (AGE) and its receptor (RAGE) in streptozotocin-induced diabetes. To induce intimal hyperplasia, autologous external jugular vein was grafted into the infrarenal abdominal aorta in 52 male Sprague-Dawley rats. In diabetic rats, distilled water with or without aminoguanidine was administrated, whereas nondiabetic rats were given distilled water alone. Vein grafts were harvested at 1 and 4 weeks after surgery for morphological analysis and semiquantitative reverse transcriptase polymerase chain reaction analysis for RAGE and nuclear factor kappaB (NF-kappaB) p65. Serum AGE level was determined by fluorospectrophotometry. Compared to nondiabetic rats, serum levels of AGE in diabetic rats administrated distilled water were significantly increased. The expression of RAGE and NF-kappaB p65, the ratio of intima to media area, and the percentage of proliferating cell nuclear antigen (PCNA)-positive cells were significantly increased in the vein graft. In diabetic rats treated with aminoguanidine, serum AGE level NF-kappaB p65 expression, the ratio of intima to media area, and the percentage of PCNA-positive cells in the vein graft were all significantly decreased. However, no difference in the expression of RAGE was found compared to the diabetic group given distilled water. Our data suggest that AGE-RAGE may play a key role in venous intimal hyperplasia in diabetes mellitus and aminoguanidine suppressed intimal hyperplasia by inhibiting this pathway.

Long-term engraftment of bone marrow-derived cells in the intimal hyperplasia lesion of autologous vein grafts.

Intimal hyperplasia of autologous vein grafts is a critical problem affecting the long-term patency of many types of vascular reconstruction. Within intimal hyperplasia lesions, smooth muscle cells are a major component, playing an essential role in the pathological process. Given that bone marrow-derived cells may differentiate into smooth muscle cells in the neointima of injured arteries, we hypothesized that the bone marrow may serve as a source for some of the smooth muscle cells within intimal hyperplasia lesions of vein grafts. To test this hypothesis, we used an established mouse model for intimal hyperplasia in wild-type mice that had been transplanted with bone marrow from a green fluorescent protein (GFP+/+) transgenic mouse. High-resolution confocal microscopy analysis performed 2 and 8 weeks after grafting demonstrated expression of GFP in 5.4 +/- 0.8% and 11.9 +/- 2.3%, respectively, of smooth muscle cells within intimal hyperplasia lesions. By 16 weeks, GFP expression in smooth muscle cells was not detected by immunohistochemistry; however, real-time PCR revealed that 20.2 +/- 1.7% of the smooth muscle cells captured from the neointima lesion by laser capture microdissection at 16 weeks contained GFP DNA. Our results suggest that bone marrow-derived cells differentiated into smooth muscle cells within the intimal lesion and may provide a novel clinical approach for decreasing intimal hyperplasia in vein grafts.

A novel mouse model of autologous venous graft intimal hyperplasia.

BACKGROUND: To investigate the molecular mechanism of autologous venous graft intimal hyperplasia, a mouse model is needed. Currently only vein to carotid artery mouse models are available and are hampered by a high thrombosis rate. We hypothesized that operating on the aorta would lead to intimal hyperplasia with decreased risk of thrombosis. MATERIALS AND METHODS: In C57BL/6J mice, the left external jugular vein was grafted into the infrarenal abdominal aorta by end-to-end anastomosis with 11-0 Ethilon. Grafts harvested at 1, 2, 4, 8, and 16 weeks postoperatively were subjected to histological and immunohistochemical analysis. RESULTS: Thirty-one of 35 mice survived; 2 mice were sacrificed secondary to thrombosis. The percentage lumen narrowing (+/-SE) was 7.8 +/- 0.3, 16.4 +/- 0.9, 19.2 +/- 0.9, 22.3 +/- 0.8, and 23.9 +/- 1.6% at 1, 2, 4, 8 and 16 weeks, respectively. Nuclear density decreased with each successive time point. The percentage of alpha-smooth-muscle actin-positive cells within the neointima peaked at 16 weeks (53%), and the percentage of cells positive for proliferating cell nuclear antigen peaked at 2 weeks (39%). CONCLUSIONS: We thus report on a novel mouse model of intimal hyperplasia in autologous venous grafts with a low thrombosis rate. Further studies using this model, coupled with genetic and bone marrow transplantation mouse models, should lead to significant enhancement in understanding of the mechanism of intimal hyperplasia.