Aya Barzelay

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.

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.

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.

Influence of Non-Toxic Doses of Bevacizumab and Ranibizumab on Endothelial Functions and Inhibition of Angiogenesis

Purpose: Ranibizumab (Lucentis) is an antibody fragment developed against all fragments of vascular endothelial growth factor (VEGF) that was approved by the FDA for treating age-related macular degeneration (AMD). Bevacizumab, a full-length anti-VEGF antibody approved for use in colon cancer, is non-FDA approved at this time but it is widely used for treating AMD. The purpose of this study was to compare the influence of Bevacizumab and Ranibizumab on angiogenesis in an in vitro model. Methods: A model consisting of H5V cells derived from murine hearts capillary endothelial cells (ECs) was used. The H5V cells were treated with three concentrations of Bevacizumab and Ranibizumab (0.125 mg/mL, 0.25 mg/mL, and 0.50 mg/mL) for 24 hr before all experiments. The effects of Bevacizumab and Ranibizumab on EC proliferation were compared by 3H-thymidine incorporation essay. Toxic effects and the safety of each drug in clinical concentrations were assessed by annexin 5 staining. The effects of the drugs on ECs functions were assessed by their ability to adhere to fibronectin and by evaluation of the cells’ tube formation capacity on matrigel. Results: Both Bevacizumab and Ranibizumab equally suppressed the adhesive properties of ECs to fibronectin, and similarly inhibited ECs’ proliferation capacity in a dose-dependent manner. Both Bevacizumab and Ranibizumab inhibited the ECs’ tube formation capacity on matrigel, and were equally safe. Conclusions: Ranibizumab and Bevacizumab at low, non-toxic doses similarly inhibit several properties of the angiogenesis process. Inhibition of ECs adhesion to fibronectin and tube formation capacity does not seem to be directly related to the anti-angiogenic effects as indicated by inhibition of VEGF. Further studies for delineating the exact mechanism of action of Ranibizumab and Bevacizumab in angiogenesis are warranted.

Power-Assisted Liposuction Versus Tissue Resection for the Isolation of Adipose Tissue–Derived Mesenchymal Stem Cells: Phenotype, Senescence, and Multipotency at Advanced Passages

BACKGROUND Adipose tissue-derived mesenchymal stem cells (ASCs) can be isolated from subcutaneous fat harvested by tissue resection or liposuction. OBJECTIVES The authors compared ASCs isolated by tissue resection or power-assisted liposuction (PAL) to determine whether either surgical procedure yielded ASCs with improved purity and competence that was preserved for several passages. METHODS For this experimental study, ASCs were isolated from fat harvested by tissue resection or PAL from six patients who underwent abdominoplasty. ASCs were counted to determine cell yields, and viabilities were assessed with an amine-reactive dye and by fluorescence-activated cell sorting (FACS). Cell phenotypes were determined by immunostaining and FACS, and doubling times were calculated. Senescence ratios of the cells were detected by gene profiling and by assaying β-galactosidase activity. Multipotency was evaluated by induced differentiation analyses. RESULTS No significant differences were observed in cell numbers or viabilities of ASCs isolated following either surgical method of fat harvesting. Both populations of cultured ASCs expressed markers of mesenchymal stem cells and preserved this expression pattern through the third passage. PAL and tissue resection yielded ASCs with similar division rates, similar senescence ratios into the fourth passage, and similar capacities to differentiate into osteocytes or adipocytes. CONCLUSIONS Fat harvested by PAL or tissue resection yielded uniform cultures of ASCs with high division rates, low senescence ratios, and multipotency preserved into passages 3 and 4. Because PAL is less invasive, it may be preferable for the isolation of ASCs.

Vitrectomy in patients 85 years of age and older: surgical outcomes and visual prognosis

Purpose To evaluate visual and surgical outcomes in very elderly patients (above 85 years of age) undergoing pars plana vitrectomy (PPV). Patients and methods A single-center, retrospective study was carried out on the medical records of 82 patients aged 85 years and older who had undergone PPV from 2006 to 2013. Patients ranged in age from 86 to 99 years, with a mean age of 88.9 years (±2.88). Visual results and intraoperative and postoperative complications were the main outcome measures. Visual improvement/worsening was defined as at least ±0.1 logMAR change. Results Mean follow-up was 7.25 months (±5.35), with a range of 1–28 months. General anesthesia was used in 63% of the operations. The most common indication was retinal detachment (27%). The ocular condition necessitating PPV was secondary to trauma (most commonly after a fall) in 10 eyes (12%). Mean visual acuity (VA) improved from 1/58 preoperatively to 1/29 at the final evaluation (p=0.014). Mean improvement in VA in eyes of patients with the comorbidity of age-related macular degeneration (n=34) was 41% lower compared to eyes of patients without the disease (n=48, p=0.013). In the subgroup of patients operated on for retinal detachment, 45.4% did not reach primary anatomic success and 45.4% needed additional retina-affecting surgery. One or more major ocular complications were reported in 24 eyes (29%), while 19 eyes (23%) had minor ocular complications. Conclusion Improved VA was documented in more than half of the older adults aged 85–99 undergoing vitrectomy. Despite the rate of complications in the very elderly, the possibility of optimizing visual function may positively affect quality of life in this subgroup.