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Rationale: Ischemic mitral regurgitation, a complication after myocardial infarction (MI), induces adaptive mitral valve (MV) responses that may be initially beneficial but eventually lead to leaflet fibrosis and MV dysfunction. We sought to examine the MV endothelial response and its potential contribution to ischemic mitral regurgitation., Objective: Endothelial, interstitial, and hematopoietic cells in MVs from post-MI sheep were quantified. MV endothelial CD45, found post MI, was analyzed in vitro., Methods and Results: Ovine MVs, harvested 6 months after inferior MI, showed CD45, a protein tyrosine phosphatase, colocalized with von Willebrand factor, an endothelial marker. Flow cytometry of MV cells revealed significant increases in CD45 + endothelial cells (VE-cadherin + /CD45 + /α-smooth muscle actin [SMA] + and VE-cadherin + /CD45 + /αSMA- cells) and possible fibrocytes (VE-cadherin - /CD45 + /αSMA + ) in inferior MI compared with sham-operated and normal sheep. CD45 + cells correlated with MV fibrosis and mitral regurgitation severity. VE-cadherin + /CD45 + /αSMA + cells suggested that CD45 may be linked to endothelial-to-mesenchymal transition (EndMT). MV endothelial cells treated with transforming growth factor-β1 to induce EndMT expressed CD45 and fibrosis markers collagen 1 and 3 and transforming growth factor-β1 to 3, not observed in transforming growth factor-β1-treated arterial endothelial cells. A CD45 protein tyrosine phosphatase inhibitor blocked induction of EndMT and fibrosis markers and inhibited EndMT-associated migration of MV endothelial cells., Conclusions: MV endothelial cells express CD45, both in vivo post MI and in vitro in response to transforming growth factor-β1. A CD45 phosphatase inhibitor blocked hallmarks of EndMT in MV endothelial cells. These results point to a novel, functional requirement for CD45 phosphatase activity in EndMT. The contribution of CD45 + endothelial cells to MV adaptation and fibrosis post MI warrants investigation., (© 2016 American Heart Association, Inc.)

A peptide analog, 4-fluorobenzoyl-RR-(L-3-(2-naphthyl)alanine)-CYEK-(L-citrulline)-PYR-(L-citrulline)-CR, covalently linked to a phospholipid, was used for targeting a lipid-based gene delivery vehicle to CXCR4(+)-cells. Characterization of transfection activity was done in vitro using a transformed rat glioma cell line (RG2) that expresses CXCR4. The substitution of the targeting lipid at increasing mole percentages in the place of helper lipids yielded a progressive increase in reporter gene expression, reaching a maximum of 2.5 times the control value at 20 mol% of ligand. The substitution of helper lipids with cysteine-derivatized phospholipid analog or phosphatidylethanolamine resulted in a progressive decrease in transfection activity, with complete inactivation of the complex occurring at 20 mol%. A DNA dose-response with 10 mol% of lipopeptide reduced the effective DNA dose at least fivefold with regard to the number of transfected cells and >20-fold with regard to the amount of gene expression. Gene transfer to rat endothelial cells was studied in the context of an arterial organ culture. Mesenteric arteries were cannulated and maintained in culture for up to 4 days. CXCR4 cell-surface expression on endothelial cells was induced after overnight incubation with vascular endothelial growth factor (VEGF). Gene transfer studies showed that only the peptide-targeted lipoplexes transfected the endothelium, and only after CXCR4 had been induced with VEGF. These results demonstrate that non-viral transfection complexes can be targeted to cells expressing CXCR4, and that gene transfer is dependent upon cell surface receptor expression levels.

We asked whether the hypoxia-regulated factor, insulin-like growth factor binding protein-3 (IGFBP3), could modulate stem cell factor receptor (c-kit+), stem cell antigen-1 (sca-1+), hematopoietic stem cell (HSC), or CD34+ endothelial precursor cell (EPC) function. Exposure of CD34+ EPCs to IGFBP3 resulted in rapid differentiation into endothelial cells and dose-dependent increases in cell migration and capillary tube formation. IGFBP3-expressing plasmid was injected into the vitreous of neonatal mice undergoing the oxygen-induced retinopathy (OIR) model. In separate studies, GFP-expressing HSCs were transfected with IGFBP3 plasmid and injected into the vitreous of OIR mice. Administering either IGFBP3 plasmid alone or HSCs transfected with the plasmid resulted in a similar reduction in areas of vasoobliteration, protection of the developing vasculature from hyperoxia-induced regression, and reduction in preretinal neovascularization compared to control plasmid or HSCs transfected with control plasmid. In conclusion, IGFBP3 mediates EPC migration, differentiation, and capillary formation in vitro. Targeted expression of IGFBP3 protects the vasculature from damage and promotes proper vascular repair after hyperoxic insult in the OIR model. IGFBP3 expression may represent a physiological adaptation to ischemia and potentially a therapeutic target for treatment of ischemic conditions.

Insulin-like growth factor-I (IGF-I) and its receptor (IGF-IR) are essential for normal ocular development and are expressed in numerous ocular cell types including lens epithelial cells, retinal pigment epithelial cells, Müller cells and endothelial cells. Endothelial cell proliferation is a common feature of proliferative retinopathies and involves abnormal growth of blood vessels within and on the surface of the retina. In an effort to inhibit the formation of these aberrant blood vessels, we cloned an IGF-IR ribozyme into an expression vector that limits expression of the ribozyme to proliferating endothelial cells. An endothelin enhancer and Cdc6 promoter chimera drives expression of the IGF-IR ribozyme. This promoter limited retinal expression of the reporter gene to proliferating endothelial cells in two mouse models of proliferative retinopathy. In addition, expression of the IGF-IR ribozyme by this promoter inhibited aberrant retinal angiogenesis in both models while preserving normal vessels. These results demonstrate the feasibility of IGF-IR ribozyme expression in a selective manner for safer treatment of abnormal angiogenesis associated with retinopathy.

Background: Systemic administration of non-viral gene therapy provides better access to tumors than local administration. Development of a promoter that restricts expression of cytotoxic proteins to the tumor vasculature will increase the safety of the system by minimizing expression in the non-dividing endothelial cells of the vasculature of non-target tissues., Methods: Cell cycle promoters were tested for selective expression in dividing cells vs. non-dividing cells in vitro and promoter strength was compared to the cytomegalovirus (CMV) promoter. Successful promoter candidates were tested in vivo using two proliferating endothelium mouse models. Ovarectomized mice were injected with estradiol prior to lipoplex administration and expression levels were measured in the lungs and uterus 4 days after administration. The second model was a subcutaneous tumor model and expression levels were measured in the lungs and tumors. For both animal models, expression levels from the proliferating endothelium promoter were compared to that obtained from a CMV promoter., Results: The results showed that the Cdc6 promoter yielded higher expression in proliferating vs. non-proliferating cells. Secondly, promoter strength could be selectively increased in endothelial cells by the addition of a multimerized endothelin enhancer (ET) to the Cdc6 promoter. Thirdly, comparison of expression levels in the lungs vs. uterus in the ovarectomized mouse model and lungs vs. tumor in the mouse tumor model showed expression was much higher in the uterus and the tumor than in the lungs for the ET/Cdc6 promoter, and expression levels were comparable to that of the CMV promoter in the hypervascularized tissues., Conclusions: These results demonstrate that the combination of the endothelin enhancer with the Cdc6 promoter yields selective expression in proliferating endothelium and can be used to express cytotoxic proteins to treat vascularized tumors., (Copyright 2006 John Wiley & Sons, Ltd.)

The purpose of this research is to develop ligand-targeted plasmid based gene delivery systems for gene transfer to tumor endothelium. Cell adhesion assays were used to test the peptide inhibition of human endothelial cell adsorption to vitronectin-treated tissue culture plates. A series of RGD containing peptides were tested in linear form and with one and two disulfide bonds. The linear and two disulfide bond peptides yielded similar IC50 (approximately 1 x 10(-7) M). Substitution of two methionines for cysteines yielded a single disulfide bond that increased the IC50 by 10-fold. The single and double disulfide peptides were derivatized to N-succinyl-dioleoylphopsphatidylethanolamine and incorporated into 100 nm liposomes radiolabeled with H-cholesterylhexadecylether. Liposome uptake by human umbilical vein endothelial cells was tested as a function of lipopeptide surface density. Increase in membrane surface density from 5 to 20mol% increased human umbilical derived endothelial cell (HUVEC) uptake of the liposomes for both the single and double disulfide peptides. Liposome uptake by HUVECs was 3-fold greater for the double disulfide compared to the single disulfide. The single and double disulfide lipopeptides were then tested for gene transfer to HUVECs using DOTMA:Cholesterol cationic liposomes. The polyplexes were formed by rapidly mixing plasmid DNA with DOTMA:CHOL liposomes at a 3:1 charge ratio in 2% ethanol, 10% lactose. The ethanol was removed by lyophilization and upon rehydration, the lipoplexes had a mean diameter of approximately 100nm. HUVEC transfection studies showed that increasing the mol% of the single disulfide RGD lipopeptide to 20mol% increased gene transfer by 10-fold. This increase in transfection could be reduced to that obtained in the absence of lipopeptide by co-incubating the HUVECs with a 100-fold excess of the single disulfide RGD peptide, thus demonstrating lipopeptide mediated gene transfer to endothelial cells.