Sofia Maysel-Auslender

Clopidogrel attenuates atheroma formation and induces a stable plaque phenotype in apolipoprotein E knockout mice

AIM Clopidogrel is a widely used anti-thrombotic for the prevention of stent thrombosis and cardiovascular events in patients with coronary atherosclerosis. Clopidogrel has been shown to exhibit anti-inflammatory effects that are related to the attenuated activation of platelets. Atherosclerosis is a complex process in which the immune system and the endothelium appear to play a prominent role. Herein, we tested the hypothesis that clopidogrel will influence plaque size and composition in the atherosclerosis prone apolipoprotein E knockout (apoE KO) mouse model. METHODS AND RESULTS Eight week old mice were fed daily with either PBS, 1 mg or 2 mg of clopidogrel for 10 weeks. Plaque size was evaluated in the aortic sinus and cellular and humoral responses were studied as well as splenic and bone marrow endothelial progenitors by FACS. Treatment with either 1 mg and 2 mg of clopidogrel significantly reduced plaque size and augmented its stability by increasing atheromatous fibrous area. Whereas antigen specific oxLDL immune response was not influenced by clopidogrel feeding, the number of atheroprotective regulatory CD4+CD25+ T cells was significantly increased. Moreover, clopidogrel treatment resulted in a prominent rise in splenic but not bone marrow derived Sca-1+/flk-1+ endothelial progenitors. CONCLUSION Clopidogrel significantly reduces atheroma burden and stabilizes aortic sinus plaques in apoE KO mice. These effects may partially be mediated by upregulation of the regulatory T cell pool and splenic endothelial progenitor cells. These findings may expand the potential applications of clopidogrel in human subjects.

HIF-1α Overexpression and Experimental Murine Atherosclerosis

Background—Lymphocytes play an important role in the progression of atherosclerosis. Recently, hypoxia inducible factor-1 (HIF-1) was found to attenuate inflammation by regulating T cell activation and cytokine production. We studied the effects of overexpression of HIF-1&agr; in ApoE knockout murine lymphocytes, on experimental atherosclerosis. Methods and Results—ApoE−/− mice were submitted to intravenous hydrodynamic injection of empty plasmid or HIF-1&agr;P564A (HIF-1&agr; mutated stabilized construct). Robust expression of HIF-1&agr; was evident in spleen cells of recipient animals. Increased expression of IL-10 as well as decreased expression of IFN-&ggr; was measured in splenocytes of HIF-1&agr;-treated mice by RT-PCR. One week postinjection, antibody array analysis revealed a pattern consistent with a T helper 1 to T helper 2 shift. On sacrifice, assessment of aortic sinus lesions revealed a significant reduction in plaque size in HIF-1&agr; injected mice. A reduced expression of IFN-&ggr; was evident in CD4+ spleen-derived lymphocytes and aortas of HIF-1&agr;-injected mice. Conclusions—HIF-1&agr; expression in mouse lymphocytes is associated with a reduced IFN-&ggr; expression and attenuation of experimental atherosclerosis.

Circulating Apoptotic Progenitor Cells A Novel Biomarker in Patients With Acute Coronary Syndromes

Background—Progenitor CD34 cells are capable of differentiating into endothelial cells and play a role in neoangiogenesis. Circulating CD34+ cells and endothelial progenitor cells are increased in acute coronary syndrome (ACS) patients possibly because of peripheral mobilization. We tested the hypothesis that circulating apoptotic progenitors are detectable in healthy subjects and altered in ACS patients. Methods and Results—Peripheral blood mononuclear cells were isolated by Ficoll density gradient from 53 patients with ACS undergoing coronary angiography and 27 healthy subjects. Apoptosis in progenitor CD34+ cells was assessed using the Annexin V-PE/7-AAD detection kit, and fluorescence-activated cell sorter analysis was performed with triple staining for CD34, annexin-V, and 7-AAD. The percentage of apoptotic CD34+ progenitors was determined in the 2 subject groups and correlated with clinical characteristics. The percentage of apoptotic CD34+ progenitor cells was significantly increased in patients with ACS as compared with healthy subjects and was associated with the extent of coronary stenosis by angiography. There was no significant correlation between apoptotic progenitor CD34+ cells and the other parameters that we examined (age, smoking, hypertension, hyperlipidemia, diabetes mellitus, ejection fraction, creatinine levels, or taking any of the various medications, including beta blockers, thiazides, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, calcium blockers, nitrates, or statins). Conclusion—We established for the first time to our knowledge an assay to detect circulating apoptotic progenitor cells using fluorescein isothiocyanate–anti-CD34 MAb, annexin V-PE, and 7-AAD and found that apoptotic CD34+ cells are increased in ACS patients and in patients with more extensive coronary artery disease. This novel assay may shed new light on the factors governing the hemeostasis of progenitor CD34+ cells.

Constitutive Expression of HIF‐1α and HIF‐2α in Bone Marrow Stromal Cells Differentially Promotes Their Proangiogenic Properties

Bone marrow stromal cells (BMSCs) contain progenitors capable of participating in postnatal angiogenesis. Hypoxia‐inducible factors (HIFs) mediate endothelial activation by driving the expression of multiple angiogenic factors. We explored the potential of HIF‐1α and HIF‐2α modification in BMSCs, as a tool to improve cell‐based angiogenic therapy. BMSCs were retrovirally transduced to express stable forms of HIF‐1α and HIF‐2α. HIF‐1α and, to a greater extent, HIF‐2α overexpression promoted differentiation of BMSCs to the endothelial lineage, evident by CD31 and Tie‐2 expression and improved adhesive properties. Whereas chemotaxis toward stromal‐derived factor 1 was higher in both HIF‐α‐expressing BMSCs, enhanced migration toward vascular endothelial growth factor was found only following overexpression of HIF‐2α, supported by a robust expression of its receptor, Flk‐1. HIF‐α expression was associated with upregulation of angiogenic proteins and improved tube formation. Cytokine arrays of endothelial cells stimulated by medium collected from HIF‐α‐expressing BMSCs revealed further angiogenic activation and improved adhesive capacity. Eventually, delivery of HIF‐2α‐transduced BMSCs induced a more robust angiogenic response, compared with sham‐transduced or HIF‐1α‐transduced BMSCs in the corneal micropocket angiogenesis model. Our results support the use of HIF‐α genes, particularly HIF‐2α, to augment the efficacy of future cell‐based therapy.

Circulating Apoptotic Progenitor Cells in Patients with Congestive Heart Failure

Background Circulating CD34+ endothelial progenitor cells (EPCs) are capable of differentiating into mature endothelial cells to assist in angiogenesis and vasculogenesis. We sought to quantify the numbers of apoptotic progenitors in patients with congestive heart failure. Methods and Results Peripheral blood mononuclear cells were isolated by Ficoll density-gradient from 58 patients with various degrees of heart failure and 23 matched controls. Apoptosis in progenitor CD34+ cells was assessed using the Annexin V-PE/PI detection kit, and FACS analysis was performed with triple staining for CD34, annexin-V and propidium iodide. The percentage of early and late apoptotic progenitor cells was determined in the subject groups and was correlated with clinical characteristics. While there was no significant difference in total CD34 positive cells or early apoptotic progenitors between control subjects and CHF patients (p = 0.42) or between severe and mild/moderate CHF groups (p = 0.544), there was an elevated number of late apoptotic progenitors in the severe CHF group compared with the mild/moderate CHF group (p =  0.03). Late apoptotic progenitors were significantly increased in CHF patients as compared to matched controls. There was also an inverse correlation between late apoptotic progenitors and ejection fraction (r = −0.252, p = 0.028) as well as a positive association with NYHA class (r = 0.223, p = 0.046). Conclusion Severe heart failure patients exhibited higher numbers of late apoptotic progenitors, and this was positively associated with NYHA class and negatively correlated with ejection fraction. This finding may shed light on the numerous factors governing the pathophysiology of CHF.

A potential role for islet-1 in post-natal angiogenesis and vasculogenesis

The LIM-homeobox transcription factor islet-1 (Isl1) marks a cell population which gives rise to myocardial, pacemaker, endothelial and smooth muscle cells, which are derived from the secondary heart field during heart embryogenesis. Isl1+ precursors have the potential of self-renewal and differentiation into endothelial, cardiomyocyte and smooth muscle lineages. The primary objective of this study was to determine whether retroviral gene delivery of Isl1 to endothelial cells and mesenchymal stem cells (MSCs) could promote angiogenic and vasculogenic properties. To this end, endothelial cells and rat MSCs were retrovirally transduced to express Isl1. Isl1 expression in endothelial cells resulted in enhanced proliferation and adhesion to fibronectin. In addition, increased IL-1b and VEGF secretion was evident in Isl1 transduced endothelial cells, concomitant with increased migratory and tube formation properties of the endothelial cells. Isl1 expression in MSCs promoted their vasculogenic properties and resulted in enhanced in vitro tube formation. Finally, Isl1 expressing endothelial cells induced enhanced in vivo vascularisation in C57BL/6J mice. These data suggest, for the first time, that Isl1 promotes postnatal angiogenesis and vasculogenesis by improving the angiogenic properties of endothelial cells and MSCs.

The 106b∼25 microRNA cluster is essential for neovascularization after hindlimb ischaemia in mice

AIMS MicroRNAs (miRNAs, miR) are endogenous short RNA sequences that regulate a wide range of physiological and pathophysiological processes. Several miRNAs control the formation of new blood vessels either by increasing or by inhibiting angiogenesis. Here, we investigated the possible role of the miR-106b∼25 cluster in postnatal neovascularization and in regulation of the angiogenic properties of adult bone marrow-derived stromal cells. METHODS AND RESULTS To study the effect of miR-106b∼25 deletion on neovascularization, we used a miR-106b∼25 knockout (KO) mouse model. After inducing hindlimb ischaemia, we showed that vascularization in ischaemic mice devoid of miR-106b∼25 is impaired, as evident from the reduced blood flow on laser Doppler perfusion imaging. The miR-106b∼25 cluster was also shown here to be an essential player in the proper functioning of bone marrow-derived stromal cells through its regulation of apoptosis, matrigel tube formation capacity, cytokine secretion, and expression of the stem-cell marker Sca-1. In addition, we showed that capillary sprouting from miR-106b∼25 KO aortic rings is diminished. CONCLUSION These results show that the miR-106b∼25 cluster regulates post-ischaemic neovascularization in mice, and that it does so in part by regulating the function of angiogenic bone marrow-derived stromal cells and of endothelial cells.

Accelerated Renal Fibrosis in Cardiorenal Syndrome Is Associated with Long-Term Increase in Urine Neutrophil Gelatinase-Associated Lipocalin Levels

Background: Cardiac events are the main cause of death among patients with end-stage renal failure. Even a mild renal disease is currently considered a major risk factor for cardiovascular complications following myocardial infarction (MI). The aim of the present study was to detect histological, sera and urine characteristics of kidney injury in cardiorenal syndrome (CRS) compared to chronic kidney disease (CKD) with an intact cardiac function. Methods: We employed a rat model for CRS, in which an acute MI (AMI) was induced 4 weeks after establishment of subtotal nephrectomy. Four weeks later, left ventricular function was assessed by echocardiography and changes in renal performance were examined using histological and biochemical parameters. Results: Increased interstitial fibrosis as well as renal inflammation were observed in renal sections derived from CRS rats, compared to subtotal nephrectomy (CKD)-only animals. Moreover, we found that even though AMI on the background of CKD was not associated with a further decrease in creatinine clearance or a further increase in sera BUN levels compared to CKD only, a significant long-term elevation in urine neutrophil gelatinase-associated lipocalin (Ngal) levels was detectable post-MI induction. Conclusions: AMI in the CKD setting is associated with accelerated renal fibrosis and long-term elevated urine Ngal values, suggesting that cardiac dysfunction contributes to accelerated intrinsic kidney injury in CKD. The data indicate that elevated urine Ngal may potentially serve as an early non-invasive laboratory parameter for a left ventricular dysfunction-related renal injury.

Ras inhibition attenuates myocardial ischemia-reperfusion injury

Myocardial injury, developed after a period of ischemia/reperfusion (I/R) results in the destruction of functional heart tissue, this being replaced by scar tissue. Intracellular signaling pathways mediating cardiomyocyte death are partially understood and involve the activation of Ras. p38-MAPK, JNK and Mst-1 are downstream effectors of Ras protein. We hypothesized that S-farnesylthiosalicylic acid (FTS), a synthetic small molecule that detaches Ras from the inner cell membrane, consequently inhibiting Ras activity, reduces I/R myocardial injury in vitro and in vivo. Wistar rat hearts were isolated, mounted on the Langendorff apparatus and subjected to ischemia (30 min, 37 degrees C) and reperfusion. During the reperfusion period, the hearts were perfused with FTS (1 microM) solution or control buffer. Left anterior descending (LAD) ligation and subsequent reperfusion was performed in two groups of Wistar rats. Rats received 5mg/kg FTS or PBS according to two protocols: (A) FTS or PBS were administered daily 7 days prior, immediately before and 14 days (every other day) after LAD occlusion or (B) every other day for 14 days post-I/R. Hearts from FTS-treated rats (Langendorff) and FTS-treated rats (protocol A) showed a significant improvement in myocardial performance and smaller scar tissue compared with the PBS group. Infarct size in the FTS-treated group was 12.7+/-2% vs. 23.7+/-4% in the PBS-treated (in vitro) group and 17.3+/-2.5% vs. 36+/-7% compared with control I/R rats (in vivo) p<0.05. These effects may be associated with the down regulation of JNK as a short-term effector and with Mst-1 in the long-term remodeling process.

Hypoxia-Inducible Factor-1α and -2α Additively Promote Endothelial Vasculogenic Properties

Objective: While both play a role in the transcriptional response of hypoxic endothelial cells (ECs), hypoxia-inducible factor-1α (HIF-1α) and HIF-2α differ in their transactivation sites, pointing at potentially different target genes. We studied the discrete and common effects of HIF-1α and HIF-2α on the cytokine expression and vasculogenic properties of ECs. Methods and Results: H5V and bovine aortic ECs were transfected to express HIF-1α, HIF-2α or both. Overexpression of HIF-1α or HIF-2α and, to a greater extent, cotransfection of HIF-1α and HIF-2α resulted in EC activation, as revealed by analysis of the adhesion capacities and adhesion molecule surface expression of ECs. From the paracrine aspect, conditioned medium from HIF-expressing ECs was found to promote the migration and tube formation capacity of wild-type ECs, mostly following HIF-1α and HIF-2α coexpression. Antibody arrays revealed altered expression of multiple cytokines, pointing at consistent additive effects of HIF-1α and HIF-2α on angiogenic protein expression. Finally, HIF-1α and HIF-2α additively promoted vessel formation in vivo, as demonstrated by a Matrigel angiogenesis assay. Conclusion: Our results further clarify the functional roles of HIF-1α and HIF-2α in ECs and for the first time demonstrate a common contribution of HIF-1α and HIF-2α to vasculogenesis.