Ira Herman - US grants

The cellular and molecular factors regulating blood flow during human brain development or in association with hypertension, infarction and stroke are not well-studied. Our five year plan will reveal whether pericytes (PC), in association with vascular endothelial cells (EC), control microvascular tone using contractile and cytoskeletal proteins. We will use cytoskeletal-specific markers in conjunction with light and electron microscopy to localize PC, EC and vascular smooth muscle in ultrathin frozen and plastic-embedded thin sections prepared from the cerebral cortex, basal ganglia, thalamus, cerebellum, pons and spinal cord of spontaneously hypertensive (SHR) and normal (Wistar-Kyoto, WKY) rat brains during normal development and following the onset of cerebrovascular disease. These results will be compared to data derived from the in situ characterization of microvascular cells present in necropsy materials obtained from normal humans and the victims of infarction and stroke. Vascular cell response to injury will be studied in the SHR and WKY after middle cerebral arterial occlusion and trauma. These results will reveal PC and EC expression during the angiogenesis accompanying cerebral ischemia, inflammation and injury. SHR and WKY cerebral microvascular cells will be isolated and characterized in vitro. Requirements for PC and EC contraction will be revealed using a computer-assisted phase contrast light microscope interfaced with a time-lapse videotape recording system. Vasoactive amine modulation of SHR vs. WKY PC and EC contractile potential and protein expression in specific microvascular beds will be quantitated. Coordination of microvascular tone via EC-PC gap junctions will be examined as a function of SHR and WKY brain development and through the onset of hypertension and stroke. The results to these fundamental in situ and in vitro experiments may ultimately reveal the basic, cellular mechanisms controlling the anomalous flow of blood seen in association with human cerebrovascular disorders.

Current notions regarding the etiology and inception of atherosclerosis suggest that loss and/or injury to the arterial endothelium represent primary cellular events in the disease process. Regulation of the endothelial cell (EC) motile response to injury may ultimately determine whether the vessel wall will be repaired or vascular disease will ensue. Morphological and biochemical experiments are planned which may reveal the molecular and structural mechanisms underlying the cellular basis of intimal integrity and the motile response to injury. Specifically, molecules of the extracellular matrix (ECM), which are present within basal laminae of blood vessels, will be used as test substrates upon which large and microvascular EC will spread and migrate following mechanical denudation in vitro. The rate and extent of these EC movements will be quantitated as a function of ECM composition using a computer-assisted, time-lapse video-micrography tape recording system. Blocking and digestion of ECM components with specific antibodies and matrix-degrading enzymes may reveal which basement membrane molecules influence the EC motile response to injury. To gain insight into the EC cytoskeletal mechanisms involved in wound repair and intimal restabilization, specific contractile protein markers will be used. Fluorescent and colloidal gold-labeled antibodies will reveal the light microscopic form and detailed ultrastructural arrangement of the major EC contractile elements. Staining patterns and molecular configurations of cytoplasmic actin, myosin and vinculin will be compared, contrasted and quantitated in normal and wounded populations of EC with documented motility as a function of ECM composition. These studies may ultimately reveal how matrix molecules recruit or reorganize the cytoskeleton into specific, supramolecular arrays capable of motile force production, tension generation and wound closure in situ. Biosynthetically-labeled cytoplasms of normal and wounded EC will be fractionated into soluble and insoluble cytoskeletal parts and immunoprecipitated with monospecific contractile protein antibodies. Electrophoretic and fluorographic analyses of resting vs. injured EC may reveal if the motile wound healing response requires new contractile protein synthesis or induces alterations in their physical form and distribution. If so, the ECM influence on this biochemical response to injury will be examined. Results of these basic experiments may ultimately reveal the molecular mechanisms utilized by EC 1) for the promotion of intimal integrity and 2) to effect wound repair following injury in vivo.

While diagnostic technology affords a more-refined awareness for the clinical indication known as hypertension and cerebrovascular disease, the molecular and cellular mechanisms that underly cerebrovascular pathophysiology are ill-defined. Moreover, little is known regarding the role the microvasculature plays in the etiology of these disease processes. Morphologic, biochemical and molecular studies using specific antibody and recombinant DNA probes will be implemented in model systems to determine whether extracellular matrix-cytoskeletal interactions modulate microvascular pericyte contractility and growth or endothelial cell response to injury, events believed central to cerebrovascular disease development. The molecular and cellular composition of normal, (Wistar-Kyoto) and spontaneously-hypertensive rat brain microvessels will be studied and characterized in situ prior to, coincident with and following hypertensive-onset. Wistar-Kyoto, spontaneously-hypertensive rat microvascular cells and their extracellular matrices will be isolated and characterized in vitro. Rat brain microvascular endothelial cell-extracellular matrices will be used as substrates to study pericyte and endothelial growth or motility following injury. Blocking with antibodies or digesting extracellular matrices with enzymes will reveal the molecular domains that modulate pericyte and endothelial cell behaviors. Labeled antibodies will be used to localize the form and detailed cytoskeletal array of cells grown on these biomatrices. Brain pericytes and endothelial cells that grow or recover from injury on vascular-derived extracellular matrices will be fractionated into soluble and insoluble cytoskeletal components prior to immunoprecipitation, electrophoresis and fluorographic analyses. mRNAs encoding cytoskeletal protein isoforms and extracellular matrix molecules will be characterized by Northern blot analysis using cDNA probes. Results of these experiments will not only reveal how cytoskeletal, extracellular matrix protein and mRNA metabolism modulate cerebrovascular cell behavior, but may ultimately reveal the molecular and cellular mechanisms that control the anomalous flow of blood seen in patients with cerebrovascular disorders.

While diagnostic technology affords a more-refined awareness for the clinical indication known as hypertension and cerebrovascular disease, the molecular and cellular mechanisms that underly cerebrovascular pathophysiology are ill-defined. Moreover, little is known regarding the role the microvasculature plays in the etiology of these disease processes. Morphologic, biochemical and molecular studies using specific antibody and recombinant DNA probes will be implemented in model systems to determine whether extracellular matrix-cytoskeletal interactions modulate microvascular pericyte contractility and growth or endothelial cell response to injury, events believed central to cerebrovascular disease development. The molecular and cellular composition of normal, (Wistar-Kyoto) and spontaneously-hypertensive rat brain microvessels will be studied and characterized in situ prior to, coincident with and following hypertensive-onset. Wistar-Kyoto, spontaneously-hypertensive rat microvascular cells and their extracellular matrices will be isolated and characterized in vitro. Rat brain microvascular endothelial cell-extracellular matrices will be used as substrates to study pericyte and endothelial growth or motility following injury. Blocking with antibodies or digesting extracellular matrices with enzymes will reveal the molecular domains that modulate pericyte and endothelial cell behaviors. Labeled antibodies will be used to localize the form and detailed cytoskeletal array of cells grown on these biomatrices. Brain pericytes and endothelial cells that grow or recover from injury on vascular-derived extracellular matrices will be fractionated into soluble and insoluble cytoskeletal components prior to immunoprecipitation, electrophoresis and fluorographic analyses. mRNAs encoding cytoskeletal protein isoforms and extracellular matrix molecules will be characterized by Northern blot analysis using cDNA probes. Results of these experiments will not only reveal how cytoskeletal, extracellular matrix protein and mRNA metabolism modulate cerebrovascular cell behavior, but may ultimately reveal the molecular and cellular mechanisms that control the anomalous flow of blood seen in patients with cerebrovascular disorders.

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

Whereas it is well established that microvascular cells play crucial roles in the etiology of diabetic retinopathy and retinopathy of prematurity, the molecular and cellular mechanisms regulating disease inception remain obscure. To approach the mechanisms regulating these retinal vasoproliferative disorders, antibody and recombinant DNA probes will be used in model systems. Results will reveal the role that cell-matrix, growth factor-matrix and cell-cell interactions play in retinal microvascular differentiation and pathology. Retinal microvascular endothelial cells and pericytes isolated from developing and post-natal retinas will be cultured alone and together. Rigid or mechanically deformable substrates, which have been synthesized by retinal vascular cells and previously shown by our group to differentially regulate vascular cell growth and contractile phenotype, will be assessed. Specifically, we will characterize the molecular mechanisms regulating retinal pericyte development and differentiation as well as the retinal microvascular endothelial cell response to injury. To this end, we will use the combined approaches of: (i) antibody localization using contractile protein isoform-specific and growth factor-specific antibodies prepared and characterized in the lab, (ii) quantitative immunoprecipitation and fluorographic analysis of biolabeled subcellular fractions derived from developing, differentiated and injured cell populations, (iii) Northern blot and transcription run-off analyses of injured endothelial cells, growth-arrested and growth-stimulated pericytes purified from neonatal and adult retinas and (iv) PCR, homology probing and antibody screening of lambda Zap II cDNA libraries derived from retinal pericytes using probes designed to reveal genetic elements regulating retinal microvascular myogenic determination. Results of these interdisciplinary studies will help to determine whether growth factor-matrix, matrix-cell and/or cell-cell interactions modulate the expression of cis DNA regulatory elements and trans-acting factors responsible for controlling retinal microvascular growth, differentiation and the unmanageable vasoproliferation associated with diabetic retinopathy.

DESCRIPTION: Experimental results derived from studies described in this five year research plan should provide essential new insights into isoactin functional diversity and the molecular mechanisms regulating isoactin-based control of cell motility. Preliminary data presented in the proposal and recent published results identify a complex of proteins, b actin, ezrin and a b actin-specific binding protein named bcap73, which play a role in membrane protrusion. In specific aim 1, the investigator will use cell microinjection and fluorescence photoactivation to quantify in vivo isoactin filament dynamics/function in wild-type and mutant cell lines which are deficient or defective in b actin. To assess critically whether non-muscle isoactin filaments perform unique cell-specific functions, the investigator will introduce into cellsfunction-blocking isoactin-specific antibodies that can discriminate b vs g actin. He anticipates that the combined results of fluorescent actin isoprotein photoactivation and anti-isoactin 'loss-of-function' studies in wild type and mutant cells will reveal whether b actin filament dynamics is sufficient to drive cytoplasmic expansion at the leading edge. In specific aim 2, the investigator will test the specific hypothesis that bcap73, either alone or with ezrin, regulates b actin nucleation and filament assembly. These activities are essential for cytoplasmic remodelling during cell motility. Quantitative iso-f-actin binding assays using combinations of full length or truncated bcap73 and ezrin, will identify the domains that nucleate or cap b actin but not other actin isoforms. Data from these in vitro assays will be compared with results of experiments from two novel fluorescent-phalloidin assays designed to reveal whether isoactin polymerization kinetics and flexibility are unique; and whether bcap73 or other actin capping/severing proteins influence these behaviors. Concomitantly, they will characterize bcap73 in wild type and mutant cell lines. These results should offer novel insights into interactions between isoactin, their binding proteins, and the membrane which give rise to functional cell motility during development and disease.

DESCRIPTION (provided by applicant): Retinal microvascular morphogenesis is a complex and highly coordinated process, which occurs during embryonic development, post-natally and in association with several visually-impairing diseases, including retinopathy of prematurity (ROP), age-related macular degeneration and diabetic retinopathy. Recent work carried out in the principal investigator's laboratory has revealed that isoactin-based cytoskeletal remodeling is integral to the microvascular migration and proliferation observed during developmental and pathologic angiogenesis, including the pericyte-based remodeling seen during retinal microvascular maturation. In a focused, inter-disciplinary research plan that will take advantage of in vitro and in vivo models using a spectrum of well-established molecular, cell biology-based and molecular genetic approaches, we aim to reveal the molecular mechanisms and the isoactin-based signaling cascades regulating (i) retinal endothelial migration driving normal and pathologic angiogenesis, and (ii) pericyte-based control of endothelial proliferation and capillary contractility. Quantitative analyses of retinal microvascular endothelial cell cultures will be performed in conjunction with experiments aimed at over-expressing the novel beta-actin specific binding and filament capping protein, betacap73, discovered in the lab. To reveal the molecular mechanisms driving isoactin-based control of developmental and pathologic angiogenesis, we will take advantage of a 'two-mouse'transgenic approach, where we will specifically induce betacap73 over-expression within the post-natal vascular endothelium. These combined results, revealing alterations in endothelial motility and impaired angiogenesis, will serve to guide cDNA expression array analyses aimed at identifying key signaling effectors controlling these pivotal microvascular events. Further, to reveal the molecular signaling mechanisms regulating pericyte contractility and retinal endothelial growth, we will characterize the role that Rho GTPase family members play in signaling vascular cytoskeletal remodeling and isoactin dynamics during microvascular morphogenesis. Based on the preliminary data recently obtained and the experimental approaches proposed, we anticipate that our research plan will provide important new insights into the molecular mechanisms regulating retinal microvascular morphogenesis during normal development and in association with the pathologic angiogenesis accompanying diabetic retinopathy.

DESCRIPTION (provided by applicant): Whereas the vascular complications accompanying diabetic retinopathy have long been recognized, the molecular and cellular events regulating the initiation and progression of pathologic angiogenesis remain poorly understood. And, while recent work has revealed the important role that microvascular endothelial cell and pericyte interactions play in controlling capillary stability and permeability, the mechanisms controlling these cell-cell interactions are poorly understood. Importantly, the regulatory roles that pericytes play in controlling microvascular dynamics during the initiation of pathologic angiogenesis remain largely unknown. Indeed, recent work carried out in the principal investigator's laboratory strongly suggests that pericytes play pivotal roles in regulating the onset and progression of pathologic angiogenesis during diabetes. Central to this paradigm-shifting hypothesis are the preliminary findings that pericyte Rho GTP- and sphingosine-1 phosphate-dependent signaling control the mechano- chemical coupling required to sustain endothelial growth arrest in vivo. Experiments outlined as specific aims for this exploratory research program seek to validate this hypothesis while successful outcomes will unveil those unknown, upstream signals and downstream effectors, which control the pericyte- dependent initiating events responsible for regulating the onset of pathologic angiogenesis and proliferative diabetic retinopathy. PUBLIC HEALTH RELEVANCE: Successful outcomes of this exploratory research program will provide the critical missing information deemed essential for our understanding the molecular and cellular components that regulate the onset and progression of pathologic angiogenesis during diabetes or aging. Anticipated findings will not only enable a newfound awareness for the mechanisms controlling the pathologic progression of microvascular lesion formation in humans, but anticipated results should provide opportunities for the development of innovative anti-angiogenesis therapeutic approaches that are not currently available.

DESCRIPTION (provided by applicant): Prevalence estimates suggest that 23.6 million US residents have diabetes, and this number is predicted to double over the next thirty years. Diabetic retinopathy is the leading cause of blindness, and occurs in 100% of Type 1 and 60% of Type 2 diabetic patients within 20 years of diagnosis, and more than 12,000 patients become blind each year due to ocular complications from this disease. The underlying cause of diabetic retinopathy is damage to the retinal microvasculature from chronic hyperglycemia leading to increased vascular permeability and vascular occlusion. Recent evidence has shown that all of these events follow from the initial loss of pericytes on microvessels in the retina, which makes endothelial cells susceptible to diabetic conditions that result in the microaneurysms, venous changes, retinal capillary loss, and retinal ischemia. We propose to target the aberrant loss of pericytes by developing and evaluating a novel adult stem cell therapy that, we hypothesize, will supplement the native pericyte population and maintain microvessel homeostasis, thereby preventing the downstream effects that ultimately lead to macular edema. We have recently shown that a sub-population of adult human adipose-derived stem cells (hASCs) spontaneously differentiates into pericytes when injected in vivo, and in so doing, stabilizes the microvascular endothelium. We will test the hypothesis that cultured ASCs contain a sub-population of pericytes (or, pericyte precursors) that exhibits the intracellular signaling machinery, contractil properties, and functional abilities to diminish microvascular leakage, reduce capillary dropout, and prevent pathologic angiogenic induction and/or EC hyper-proliferation in three relevant murine models: oxygen-induced retinopathy (OIR), streptozocin induced diabetes (STZ), and Akimba whose pathological outcomes are related to early non-proliferative and late proliferative aspects of human DR. Our strategy is to supplement the endogenous pericyte population in order to enhance endothelial stability and prevent vessel loss. Therefore, we have designed three aims to evaluate the putative pericyte capacity of hASCs in the retina. Aim 1: Identify the hASC subpopulation(s) capable of becoming functional pericytes in vitro; Aim 2: Determine the capacity of hASCs to stabilize retinal vessels against acute (OIR) and chronic (STZ & Akimba) vascular insults; Aim 3: Determine the capacity of hASCs to enhance retinal revascularization and stabilize aberrant neovascularization following acute (OIR) and chronic (STZ & Akimba) vascular insults.