Abdul Q. Sheikh

Recapitulation of Developing Artery Muscularization in Pulmonary Hypertension

Excess smooth muscle accumulation is a key component of many vascular disorders, including atherosclerosis, restenosis, and pulmonary artery hypertension, but the underlying cell biological processes are not well defined. In pulmonary artery hypertension, reduced pulmonary artery compliance is a strong independent predictor of mortality, and pathological distal arteriole muscularization contributes to this reduced compliance. We recently demonstrated that embryonic pulmonary artery wall morphogenesis consists of discrete developmentally regulated steps. In contrast, poor understanding of distal arteriole muscularization in pulmonary artery hypertension severely limits existing therapies that aim to dilate the pulmonary vasculature but have modest clinical benefit and do not prevent hypermuscularization. Here, we show that most pathological distal arteriole smooth muscle cells, but not alveolar myofibroblasts, derive from pre-existing smooth muscle. Furthermore, the program of distal arteriole muscularization encompasses smooth muscle cell dedifferentiation, distal migration, proliferation, and then redifferentiation, thereby recapitulating many facets of arterial wall development.

Smooth muscle cell progenitors are primed to muscularize in pulmonary hypertension.

During the onset of pulmonary hypertension, a single specialized preexisting smooth muscle cell located at each muscularized-unmuscularized pulmonary arteriole border migrates distally and clonally expands, giving rise to the pathological distal arteriole smooth muscle. A smooth transition in pulmonary hypertension Recently, researchers sought to explain the incredible growth rate of the superheroes the Incredible Hulk and Captain America. Although no “super-soldier serum” exists, they decided it might be possible to recreate such a rapid increase in muscle mass through gene editing, but ultimately, the transformation could be “traumatic.” In a real-life situation, Sheikh and colleagues similarly investigated how the overgrowth of smooth muscle in the vasculature happens in pulmonary hypertension, with the hopes of targeting this process and preventing the onset of PH—a disease with few therapeutic options. In PH, normally unmuscularized tissue becomes muscularized, likely due to low oxygen (hypoxia). The authors used a series of genetic mouse models to characterize a unique cell type that expressed a smooth muscle cell (SMC) marker and platelet-derived growth factor receptor-β (PDGFR-β), and reside at the border zone of muscle and nonmuscle in the pulmonary arterioles. Knowing that these precursor cells are “primed” to become arteriole SMCs, Sheikh et al. tracked the cells over time, in living mice, during hypoxia-induced PH and discovered that the SMCs expanded clonally (from a single, specialized cell). Human pulmonary artery SMCs expressed high levels of a factor known as KLF4, which is downstream in PDGF signaling. Back in the mice, the authors found that KLF4 was required by the SMCs to migrate distally and “muscularize.” This novel SMC precursor therefore plays a major role in PH, and—perhaps much to the benefit of our favorite superheroes’ enemies—targeting the now-clear mechanisms of SMC migration and proliferation could prevent the characteristic muscular transition in disease. Excess and ectopic smooth muscle cells (SMCs) are central to cardiovascular disease pathogenesis, but underlying mechanisms are poorly defined. For instance, pulmonary hypertension (PH) or elevated pulmonary artery blood pressure is a devastating disease with distal extension of smooth muscle to normally unmuscularized pulmonary arterioles. We identify novel SMC progenitors that are located at the pulmonary arteriole muscular-unmuscular border and express both SMC markers and the undifferentiated mesenchyme marker platelet-derived growth factor receptor-β (PDGFR-β). We term these cells “primed” because in hypoxia-induced PH, they express the pluripotency factor Kruppel-like factor 4 (KLF4), and in each arteriole, one of them migrates distally, dedifferentiates, and clonally expands, giving rise to the distal SMCs. Furthermore, hypoxia-induced expression of the ligand PDGF-B regulates primed cell KLF4 expression, and enhanced PDGF-B and KLF4 levels are required for distal arteriole muscularization and PH. Finally, in PH patients, KLF4 is markedly up-regulated in pulmonary arteriole smooth muscle, especially in proliferating SMCs. In sum, we have identified a pool of SMC progenitors that are critical for the pathogenesis of PH, and perhaps other vascular disorders, and therapeutic strategies targeting this cell type promise to have profound implications.

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.

Tissue-engineered provisional matrix as a novel approach to enhance diabetic wound healing.

Inherent pathologies associated with diabetic wound microenvironment including increased proteolysis, inflammatory dysregulation, and impaired neovascularization prevent timely resolution of chronic diabetic ulcers. It is hypothesized that augmentation of local wound microenvironment with a stable provisional matrix formed by proteolysis‐resistant angiogenic peptide nanofibers (NFs) will create permissive environment for attenuated inflammation, enhanced neovascularization, and improved diabetic wound healing. Using murine excisional wound healing models, full‐thickness dorsal skin wounds were treated with either NFs or control solutions (phosphate buffered saline; hyaluronic acid) and analyzed for morphology, inflammatory response, neovascularization, and biomechanical properties. NF treatment of diabetic wounds stimulated formation of a robust pro‐angiogenic in situ tissue‐engineered provisional matrix leading to a significant decrease in wound inflammatory cell infiltration and proinflammatory interleukin‐6 levels, a significant increase in endothelial and endothelial progenitor cell infiltration, vascular endothelial growth factor levels, and neovascularization (day 7), as well as improved wound morphology, accelerated wound closure, and significantly stronger repair tissue (day 28). These results suggest that appropriate design of provisional matrix may compensate for some of the complex disruptions in diabetic wound microenvironment and provide missing cues to cells and direct in situ responses toward improved healing, which is promising for future development of new therapies for diabetic ulcers.

Regulation of endothelial MAPK/ ERK signalling and capillary morphogenesis by low-amplitude electric field

Low-amplitude electric field (EF) is an important component of wound-healing response and can promote vascular tissue repair; however, the mechanisms of action on endothelium remain unclear. We hypothesized that physiological amplitude EF regulates angiogenic response of microvascular endothelial cells via activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. A custom set-up allowed non-thermal application of EF of high (7.5 GHz) and low (60 Hz) frequency. Cell responses following up to 24 h of EF exposure, including proliferation and apoptosis, capillary morphogenesis, vascular endothelial growth factor (VEGF) expression and MAPK pathways activation were quantified. A db/db mouse model of diabetic wound healing was used for in vivo validation. High-frequency EF enhanced capillary morphogenesis, VEGF release, MEK-cRaf complex formation, MEK and ERK phosphorylation, whereas no MAPK/JNK and MAPK/p38 pathways activation was observed. The endothelial response to EF did not require VEGF binding to VEGFR2 receptor. EF-induced MEK phosphorylation was reversed in the presence of MEK and Ca2+ inhibitors, reduced by endothelial nitric oxide synthase inhibition, and did not depend on PI3K pathway activation. The results provide evidence for a novel intracellular mechanism for EF regulation of endothelial angiogenic response via frequency-sensitive MAPK/ERK pathway activation, with important implications for EF-based therapies for vascular tissue regeneration.

Diabetic Wound Healing In A MMP9‐/‐ Mouse Model

Reduced mobilization of endothelial progenitor cells (EPCs) from the bone marrow (BM) and impaired EPC recruitment into the wound represent a fundamental deficiency in the chronic ulcers. However, mechanistic understanding of the role of BM‐derived EPCs in cutaneous wound neovascularization and healing remains incomplete, which impedes development of EPC‐based wound healing therapies. The objective of this study was to determine the role of EPCs in wound neovascularization and healing both under normal conditions and using single deficiency (EPC) or double‐deficiency (EPC + diabetes) models of wound healing. MMP9 knockout (MMP9 KO) mouse model was utilized, where impaired EPC mobilization can be rescued by stem cell factor (SCF). The hypotheses were: (1) MMP9 KO mice exhibit impaired wound neovascularization and healing, which are further exacerbated with diabetes; (2) these impairments can be rescued by SCF administration. Full‐thickness excisional wounds with silicone splints to minimize contraction were created on MMP9 KO mice with/without streptozotocin‐induced diabetes in the presence or absence of tail‐vein injected SCF. Wound morphology, vascularization, inflammation, and EPC mobilization and recruitment were quantified at day 7 postwounding. Results demonstrate no difference in wound closure and granulation tissue area between any groups. MMP9 deficiency significantly impairs wound neovascularization, increases inflammation, decreases collagen deposition, and decreases peripheral blood EPC (pb‐EPC) counts when compared with wild‐type (WT). Diabetes further increases inflammation, but does not cause further impairment in vascularization, as compared with MMP9 KO group. SCF improves neovascularization and increases EPCs to WT levels (both nondiabetic and diabetic MMP9 KO groups), while exacerbating inflammation in all groups. SCF rescues EPC‐deficiency and impaired wound neovascularization in both diabetic and nondiabetic MMP9 KO mice. Overall, the results demonstrate that BM‐derived EPCs play a significant role during wound neovascularization and that the SCF‐based therapy with controlled inflammation could be a viable approach to enhance healing in chronic diabetic wounds.

Vascular Cells in Blood Vessel Wall Development and Disease

The vessel wall is composed of distinct cellular layers, yet communication among individual cells within and between layers results in a dynamic and versatile structure. The morphogenesis of the normal vascular wall involves a highly regulated process of cell proliferation, migration, and differentiation. The use of modern developmental biological and genetic approaches has markedly enriched our understanding of the molecular and cellular mechanisms underlying these developmental events. Additionally, the application of similar approaches to study diverse vascular diseases has resulted in paradigm-shifting insights into pathogenesis. Further investigations into the biology of vascular cells in development and disease promise to have major ramifications on therapeutic strategies to combat pathologies of the vasculature.

Cell Autonomous and Non-cell Autonomous Regulation of SMC Progenitors in Pulmonary Hypertension

SUMMARY Pulmonary hypertension is a devastating disease characterized by excessive vascular muscularization. We previously demonstrated primed platelet-derived growth factor receptor β+ (PDGFR-β+)/smooth muscle cell (SMC) marker+ progenitors at the muscular-unmuscular arteriole border in the normal lung, and in hypoxia-induced pulmonary hypertension, a single primed cell migrates distally and expands clonally, giving rise to most of the pathological smooth muscle coating of small arterioles. Little is known regarding the molecular mechanisms underlying this process. Herein, we show that primed cell expression of Kruppel-like factor 4 and hypoxia-inducible factor 1-α(HIF1-α) are required, respectively, for distal migration and smooth muscle expansion in a sequential manner. In addition, the HIF1-α/PDGF-B axis in endothelial cells non-cell autonomously regulates primed cell induction, proliferation, and differentiation. Finally, myeloid cells transdifferentiate into or fuse with distal arteriole SMCs during hypoxia, and Pdgfb deletion in myeloid cells attenuates pathological muscularization. Thus, primed cell autonomous and non-cell autonomous pathways are attractive therapeutic targets for pulmonary hypertension.

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