Pneumatic displacement with intravitreal tPA injection versus vitrectomy with sub retinal tPA injection in small and medium sub macular hemorrhages- a multicenter comparative study.

PURPOSE: Comparing between the visual outcomes and post operative complications of two surgical treatments for sub macular hemorrhage, pars plana vitrectomy with tissue plasminogen activator (tPA) injection procedure, and pneumatic displacement of submacular hemorrhage with intravitreal tPA injection. METHODS: A retrospective chart review of patients with sub macular hemorrhage (SMH) was performed. Data was collected from 150 patients with sub macular hemorrhage. Patients were followed up from the day of admission and up to a year post surgery. Evaluation included visual acuity, optical coherence tomography (OCT), fundus examination and rates of complications. RESULTS: Pars plana vitrectomy procedure has showed a better visual outcome in small SMH. Comparing complications between the two treatment modalities, no significant difference has been found in the study. CONCLUSIONS: Pars plana vitrectomy and tPA showed a clear advantage with a trend of better visual acuity as well as a significant predictor to better visual acuity for small and medium sub macular hemorrhage.

Sequential Fabrication of a Three-Layer Retina-like Structure.

Tissue engineering is considered a promising approach to treating advanced degenerative maculopathies such as nonexudative age-related macular degeneration (AMD), the leading cause of blindness worldwide. The retina consists of several hierarchical tissue layers, each of which is supported by a layer underneath. Each of these layers has a different morphology and requires distinct conditions for proper assembly. In fact, a prerequisite step for the assembly of each of these layers is the organization of the layer underneath. Advanced retinal degeneration includes degeneration of the other retina layers, including the choroid, the retinal pigmented epithelium (RPE), and the photoreceptors. Here, we report a step-by-step fabrication process of a three-layer retina-like structure. The process included the 3D printing of a choroid-like structure in an extracellular matrix (ECM) hydrogel, followed by deposition of the RPE monolayer. After the formation of the blood vessel-RPE interface, the photoreceptor cells were deposited to interact with the RPE layer. At the end of the fabrication process, each layer was characterized for its morphology and expression of specific markers, and the integration of the three-layer retina was evaluated. We envision that such a retina-like structure may be able to attenuate the deterioration of a degenerated retina and improve engraftment and regeneration. This retinal implant may potentially be suitable for a spectrum of macular degenerative diseases for which there are currently no cures and may save millions from complete blindness.

Regenerative Effect of Adipose Derived Mesenchymal Stem Cells on Ganglion Cells in the Hypoxic Organotypic Retina Culture.

Background and Objectives: To examine whether ischemic retinal ganglion cells (RGCs) will be salvaged from cell death by human adipose-derived mesenchymal stem cells (ADSCs) in an organotypic retina model. Methods and Results: Deprived of arterial oxygen supply, whole mice retinas were cultured as an ex vivo organotypic cultures on an insert membrane in a 24-well plate. The therapeutic potential of ADSCs was examined by co-culture with organotypic retinas. ADSCs were seeded on top of the RGCs allowing direct contact, or at the bottom of the well, sharing the same culture media and allowing a paracrine activity. The number of surviving RGCs was assessed using Brn3a staining and confocal microscopy. Cytokine secretion of ADSCs to medium was analyzed by cytokine array. When co-cultured with ADSCs, the number of surviving RGCs was similarly significantly higher in both treatment groups compared to controls. Analysis of ADSCs cytokines secretion profile, showed secretion of anti-apoptotic and pro-proliferative cytokines (threshold＞1.4). Transplantation of ADSCs in a co-culture system with organotypic ischemic retinas resulted in RGCs recovery. Since there was no advantage to direct contact of ADSCs with RGCs, the beneficial effect seen may be related to paracrine activity of ADSCs. Conclusions: These data correlated with secretion profile of ADSCs' anti-apoptotic and pro-proliferative cytokines.

Retinal Lineage Therapeutic Specific Effect of Human Orbital and Abdominal Adipose-Derived Mesenchymal Stem Cells.

Retinal degenerative diseases are one of the main causes of complete blindness in aged population. In this study, we compared the therapeutic potential for retinal degeneration of human mesenchymal stem cells derived from abdominal subcutaneous fat (ABASCs) or from orbital fat (OASCs) due to their accessibility and mutual embryonic origin with retinal tissue, respectively. OASCs were found to protect RPE cells from cell death and were demonstrated to increase early RPE precursor markers, while ABASCs showed a raise in retinal precursor marker expression. Subretinal transplantation of OASCs in a mouse model of retinal degeneration led to restoration of the RPE layer while transplantation of ABASCs resulted in a significant restoration of the photoreceptor layer. Taken together, we demonstrated a lineage-specific therapeutic effect for either OASCs or ABASCs in retinal regeneration.

Diagnosis of Peripheral Retinoschisis Using Ultrasound Biomicroscopy.

BACKGROUND AND OBJECTIVE: Retinal imaging can help differentiate retinoschisis (RS) from retinal detachment (RD). This study describes new sonographic features of RS using ultrasound biomicroscopy (UBM) and evaluates their diagnostic value. PATIENTS AND METHODS: Medical records of subjects diagnosed with RS and RD who underwent imaging prior to intervention were reviewed. Images were evaluated for detachment shape ultrasound (US) B-mode, as well as presence of intraretinal pillars, retinal layers split, and intraretinal cysts on UBM. RESULTS: Of 48 eyes from 48 patients in the study, 25 were diagnosed as RS and 23 as RD. "Retinal layers split" was the most common UBM finding in the RS group (72%), followed by intraretinal pillars (64%) and intraretinal cysts (36%). No RD case exhibited these findings (P < .001). CONCLUSIONS: UBM might assist in difficult cases to differentiate between RS and RD by detection of the unique sonographic features of RS described herein. [Ophthalmic Surg Lasers Imaging Retina. 2019;50:e196-e202.].

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.

Adipose-Derived Mesenchymal Stem Cells Migrate and Rescue RPE in the Setting of Oxidative Stress.

Oxidative stress leads to the degeneration of retinal pigment epithelial (RPE) and photoreceptor cells. We evaluated the potential of adipose-derived mesenchymal stem cells (ASCs) as a therapeutic tool by studying the migration capacity of ASCs in vitro and their protective effect against RPE cell death under oxidative stress in vitro and in vivo. ASCs exhibited enhanced migration when exposed to conditioned medium of oxidative stressed RPE cells obtained by hydrogen peroxide. Migration-related axis SDF-1/CXCR4 was studied, and upregulation of SDF-1 in stressed RPE and of CXCR4 in ASCs was detected. Moreover, ASCs' conditioned medium prevented H2O2-induced cell death of RPE cells. Early passage ASCs had high expression level of HGF, low VEGF levels, and unmodulated IL-1ß levels, compared to late passage ASCs. Thus, early passage ASCs show the potential to migrate towards damaged RPE cells and protect them in a paracrine manner from cell death induced by oxidative stress. In vivo, mice received systemic injection of NaIO3, and 72 h later, ASCs were transplanted in the subretinal space. Seven days after ASC transplantation, the eyes were enucleated fixed and frozen for immunohistochemical analysis. Under such conditions, ASC-treated mice showed preservation of nuclear layers in the outer nuclear layer and stronger staining of RPE and photoreceptor layer, compared to PBS-treated mice. Taken together, our results indicate that ASCs are able to home in on damaged RPE cells and protect against damage to the RPE and PR layers caused by oxidative stress. These data imply the potential that ASCs have in regenerating RPE under oxidative stress, providing the basis for a therapeutic approach to retinal degeneration diseases related to oxidative stress that could help save the eyesight of millions of people worldwide.

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 ß-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.

Islet-1 gene delivery improves myocardial performance after experimental infarction.

OBJECTIVE: The LIM-homeobox transcription factor Isl1 plays a crucial role during heart embryogenesis and later on gives rise to adult resident cardiac stem cells. In this study, we aimed to discover new extra cardiac populations of Isl1 stem cells. We then investigated endogenous Isl1 kinetics after myocardial infarction (MI), and the effect of intra-myocardial gene transfer of naked DNA encoding Isl1 on functional recovery after MI. METHODS: We used the transgenic mice Isl1/cre/Z/EG for lineage tracing of extra cardiac Isl1 stem cells. Non transgenic mice were used to study Isl1 kinetics post-MI by RT-PCR and FACS analysis. MI was induced in non transgenic mice by permanent ligation of the left anterior descending coronary artery (LAD). Naked DNA encoding Isl1 was injected to the peri-infarct region. Evaluation of cardiac performance was conducted by echocardiogram. Analysis of myocardial fibrosis and number of vessels was performed on histological cryosections. RESULTS AND CONCLUSIONS: Isl1 gives rise to subpopulations of progenitors in both the bone marrow and spleen, and is re-expressed in the spleen and left ventricle following MI. Intramyocardial gene transfer of Isl1 to the border zone of the infarcted hearts resulted in partial salvage of left ventricular function, enhanced vascularization, and reduced myocardial fibrosis. The Isl1 gene appears to be an attractive reparative target for future management of myocardial dysfunction.

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.

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.

HIF-1alpha 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-1alpha in ApoE knockout murine lymphocytes, on experimental atherosclerosis. METHODS AND RESULTS: ApoE-/- mice were submitted to intravenous hydrodynamic injection of empty plasmid or HIF-1alphaP564A (HIF-1alpha mutated stabilized construct). Robust expression of HIF-1alpha was evident in spleen cells of recipient animals. Increased expression of IL-10 as well as decreased expression of IFN-gamma was measured in splenocytes of HIF-1alpha-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-1alpha injected mice. A reduced expression of IFN-gamma was evident in CD4+ spleen-derived lymphocytes and aortas of HIF-1alpha-injected mice. CONCLUSIONS: HIF-1alpha expression in mouse lymphocytes is associated with a reduced IFN-gamma expression and attenuation of experimental atherosclerosis.

Constitutive expression of HIF-1alpha and HIF-2alpha 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-1alpha and HIF-2alpha modification in BMSCs, as a tool to improve cell-based angiogenic therapy. BMSCs were retrovirally transduced to express stable forms of HIF-1alpha and HIF-2alpha. HIF-1alpha and, to a greater extent, HIF-2alpha 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-alpha-expressing BMSCs, enhanced migration toward vascular endothelial growth factor was found only following overexpression of HIF-2alpha, supported by a robust expression of its receptor, Flk-1. HIF-alpha expression was associated with upregulation of angiogenic proteins and improved tube formation. Cytokine arrays of endothelial cells stimulated by medium collected from HIF-alpha-expressing BMSCs revealed further angiogenic activation and improved adhesive capacity. Eventually, delivery of HIF-2alpha-transduced BMSCs induced a more robust angiogenic response, compared with sham-transduced or HIF-1alpha-transduced BMSCs in the corneal micropocket angiogenesis model. Our results support the use of HIF-alpha genes, particularly HIF-2alpha, to augment the efficacy of future cell-based therapy. Disclosure of potential conflicts of interest is found at the end of this article.