Jennifer R. Hurley

REGULATION OF ENDOTHELIAL CELL ACTIVATION AND ANGIOGENESIS BY INJECTABLE PEPTIDE NANOFIBERS

RAD16-II peptide nanofibers are promising for vascular tissue engineering and were shown to enhance angiogenesis in vitro and in vivo, although the mechanism remains unknown. We hypothesized that the pro-angiogenic effect of RAD16-II results from low-affinity integrin-dependent interactions of microvascular endothelial cells (MVECs) with RAD motifs. Mouse MVECs were cultured on RAD16-II with or without integrin and MAPK/ERK pathway inhibitors, and angiogenic responses were quantified. The results were validated in vivo using a mouse diabetic wound healing model with impaired neovascularization. RAD16-II stimulated spontaneous capillary morphogenesis, and increased β(3) integrin phosphorylation and VEGF expression in MVECs. These responses were abrogated in the presence of β(3) and MAPK/ERK pathway inhibitors or on the control peptide without RAD motifs. Wide-spectrum integrin inhibitor echistatin completely abolished RAD16-II-mediated capillary morphogenesis in vitro and neovascularization and VEGF expression in the wound in vivo. The addition of the RGD motif to RAD16-II did not change nanofiber architecture or mechanical properties, but resulted in significant decrease in capillary morphogenesis. Overall, these results suggest that low-affinity non-specific interactions between cells and RAD motifs can trigger angiogenic responses via phosphorylation of β(3) integrin and MAPK/ERK pathway, indicating that low-affinity sequences can be used to functionalize biocompatible materials for the regulation of cell migration and angiogenesis, thus expanding the current pool of available motifs that can be used for such functionalization. Incorporation of RAD or similar motifs into protein engineered or hybrid peptide scaffolds may represent a novel strategy for vascular tissue engineering and will further enhance design opportunities for new scaffold materials.

Diabetes Alters Intracellular Calcium Transients in Cardiac Endothelial Cells

Diabetic cardiomyopathy (DCM) is a diabetic complication, which results in myocardial dysfunction independent of other etiological factors. Abnormal intracellular calcium ([Ca2+]i) homeostasis has been implicated in DCM and may precede clinical manifestation. Studies in cardiomyocytes have shown that diabetes results in impaired [Ca2+]i homeostasis due to altered sarcoplasmic reticulum Ca2+ ATPase (SERCA) and sodium-calcium exchanger (NCX) activity. Importantly, altered calcium homeostasis may also be involved in diabetes-associated endothelial dysfunction, including impaired endothelium-dependent relaxation and a diminished capacity to generate nitric oxide (NO), elevated cell adhesion molecules, and decreased angiogenic growth factors. However, the effect of diabetes on Ca2+ regulatory mechanisms in cardiac endothelial cells (CECs) remains unknown. The objective of this study was to determine the effect of diabetes on [Ca2+]i homeostasis in CECs in the rat model (streptozotocin-induced) of DCM. DCM-associated cardiac fibrosis was confirmed using picrosirius red staining of the myocardium. CECs isolated from the myocardium of diabetic and wild-type rats were loaded with Fura-2, and UTP-evoked [Ca2+]i transients were compared under various combinations of SERCA, sarcoplasmic reticulum Ca2+ ATPase (PMCA) and NCX inhibitors. Diabetes resulted in significant alterations in SERCA and NCX activities in CECs during [Ca2+]i sequestration and efflux, respectively, while no difference in PMCA activity between diabetic and wild-type cells was observed. These results improve our understanding of how diabetes affects calcium regulation in CECs, and may contribute to the development of new therapies for DCM treatment.

Self Assembling Peptide Nanofibers for Extracellular Matrix Remodeling in Diabetic Cardiomyopathy

Diabetes is one of the most common chronic illnesses in the world and 25.8 million individuals – 8.3% of the population– are afflicted in the United States alone with annual medical costs estimated at

Self-Assembling Peptide Nanofibers for MMP Delivery and Cardiac Regeneration in Diabetes

174 billion in 2007 [1]. While there are many life-threatening complications associated with diabetes, heart disease is one of the most severe with a higher than normal diabetes-associated death rate. Diabetic cardiomyopathy (DCM) is a diabetes-associated cardiovascular condition defined as ventricular dysfunction in the absence of other etiological factors, such as hypertension or coronary heart disease [2-4], which results in pathological alterations to the myocardium including circulatory defects, impaired heart muscle contraction, and progressive fibrosis. The elusive and poorly defined nature of DCM indicates that there exists a need for novel approaches for treatment of diabetic cardiomyopathy which may focus on alternative molecular mechanisms for the disease. Extracellular matrix (ECM) turnover and remodeling are essential in many physiological processes yet their regulation is impaired in DCM, leading to damaging structural, geometric and functional changes in the heart [2,3,5,6]. ECM turnover can be regulated by many factors, including matrix metalloproteinases (MMPs) [6], angiotensin II [7], aldosterone [4], nuclear factor kappa B (NF-κB) [8], transforming growth factor-beta 1 (TGF-β1) [9], nitric oxide [10], advanced glycation end products [5], and kinins [11]. Importantly, recent studies of diabetic human patients [12,13] and in animal models [7,9,14-16] suggest that MMP activity is impaired in diabetes, thus highlighting this particular mechanism as a novel therapeutic target. In particular, studies such as these have shown that dysregulation of cardiac MMP-2 expression contributes to the increased collagen deposition, progressive fibrosis, increased ventricular stiffness, and cardiac dysfunction seen in diabetic cardiomyopathy in rodent animal models. This MMP-2 deficiency likely stems from diabetes-related changes to the cardiac fibroblast phenotype, such as increased collagen synthesis by cardiac fibroblasts under diabetic conditions [17-19]. At the same time, studies in murine models of type II (db/db) diabetes have shown decreased MMP activation in diabetic fibroblasts, as well as impairments in vital cellular processes, including reduced growth factor expression and reduced cellular migration [20]. Therefore, overcoming the inhibitory effects of diabetic conditions on matrix remodeling by cardiac fibroblasts through stimulation of native MMP-2 expression or delivery of exogenous MMP-2 represents a novel target for therapeutic treatment of DCM.

Endothelial-Fibroblast Interactions in Angiogenesis and Matrix Remodeling

Diabetes is a serious problem in the United States, afflicting 7.8% of the population with annual medical costs estimated at

Fibroblasts Induce Mechanical Changes in the Extracellular Environment and Enhance Capillary-Like Network Formation

116B in 2007 (1). Diabetic cardiomyopathy (DCM) is a cardiovascular complication of diabetes resulting in pathological alterations to the myocardium including circulatory defects, impaired heart muscle contraction, and progressive fibrosis. Cardiac fibrosis is associated with an imbalance between the deposition of the extracellular matrix (ECM) proteins by cardiac fibroblasts and the ECM proteolytic degradation via matrix metalloproteinases (MMPs). Recent studies have demonstrated that in the diabetic heart, expression and activity of MMP-2 are reduced, resulting in increased collagen accumulation and cardiac dysfunction (2). These observations suggest that a MMP-related mechanism may contribute to cardiac fibrosis, and that it may be attenuated through stimulation of native MMP-2 expression or delivery of exogenous MMP-2. Therefore, reduced MMP-2 activity in DCM may represent a novel target for therapeutic treatment (3). To achieve this, a special proteolytically-stable delivery scaffold would be needed, because native ECM is rapidly degraded by MMPs. The goal of this study is to determine if self-assembling peptide nanofibers can be used for long-term (several weeks) MMP delivery and enhancement of cardiac remodeling. This study tests the hypothesis that increased MMP-2 concentration (native or exogenous) in the nanofiber environment will promote matrix remodeling in diabetic cardiac fibroblasts in vitro.Copyright

Complex temporal regulation of capillary morphogenesis by fibroblasts.

Revascularization is critical for successful regeneration of ischemic cardiac tissue after injury. To achieve revascularization in engineered cardiac grafts, it is necessary to understand the interactions between major cardiac cell types. The importance of cardiomyocyte-endothelial interactions in angiogenesis is well documented [1]; however, less is known about interactions between endothelial and stromal cells, fibroblasts in particular. Studies indicate that during capillary assembly, fibroblasts (FBs) provide chemical signaling via growth factor expression and endothelial activation and proliferation [2]. In addition, fibroblasts deposit extracellular matrix (ECM) proteins [3] and also play a role in matrix remodeling. The objective of our study was to further investigate the role of endothelial-fibroblast interactions in angiogenesis with a focus on FB regulation of the extracellular mechanical environment. We and others have recently shown that self-assembling peptide nanoscaffold is a promising material for cardiac tissue regeneration, enhancing angiogenesis in vitro and promoting tissue neovascularization in vivo [1, 4–5]. An important advantage of this nanoscaffold is the ability to control its material properties, such as stiffness and rate of MMP degradation, through its sequence and/or concentration [6]. In this study, RAD16-II peptide nanoscaffold was used as a controlled system to test the hypothesis that fibroblasts regulate angiogenesis via modifying the extracellular mechanical environment through mechanisms including cell-mediated scaffold disruption and matrix remodeling.Copyright

Angiogenic microenvironment augments impaired endothelial responses under diabetic conditions

Cardiac tissue engineering studies have demonstrated the importance of revascularization in engineered grafts for successful implantation and regeneration [1]. Understanding the myocardium’s complex cellular organization and the interactions between the major cardiac cell types (cardiomyocytes, endothelial cells, and cardiac fibroblasts) is critical for revascularization. Our previous studies have shown the importance of cardiomyocyte-endothelial interactions [2]. However, there is limited information available on endothelial-fibroblast interactions. We and others have previously observed that during capillary assembly, fibroblasts provide chemical signaling via expression of growth factors [3, 4]. In addition, fibroblasts may also regulate angiogenesis through alterations to the mechanical environment via myocardial remodeling, including matrix degradation and deposition, and tissue contraction. Changes to the extracellular mechanical enviroment may lead to changes in basic cell functions such as proliferation, apoptosis, and growth factor expression.Copyright