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Biomedical subjects

Robert D Simari

Publications and source records attributed to Robert D Simari.

At least 19 recordsLinked to original sources
PubMed2006

Magnetically targeted endothelial cell localization in stented vessels.

OBJECTIVES: A novel method to magnetically localize endothelial cells at the site of a stented vessel wall was developed. The application of this strategy in a large animal model is described. BACKGROUND: Local delivery of blood-derived endothelial cells has been shown to facilitate vascular healing in animal models. Therapeutic utilization has been limited by an inability to retain cells in the presence of blood flow. We hypothesized that a magnetized stent would facilitate local retention of superparamagnetically labeled cells. METHODS: Cultured porcine endothelial cells were labeled with endocytosed superparamagnetic iron oxide microspheres. A 500:1 microsphere-to-cell ratio was selected for in vivo experiments based on bromo-deoxyuridine incorporation and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assays. Stents were magnetized and implanted in porcine coronary and femoral arteries using standard interventional equipment. Labeled endothelial cells were delivered locally during transient occlusion of blood flow. RESULTS: The delivered cells were found attached to the stent struts and were also distributed within the adjacent denuded vessel wall at 24 h. CONCLUSIONS: Magnetic forces can be used to rapidly place endothelial cells at the site of a magnetized intravascular stent. The delivered cells are retained in the presence of blood flow and also spread to the adjacent injured vessel wall. Potential applications include delivering a cell-based therapeutic effect to the local vessel wall as well as downstream tissue.

Animals↗
PubMed2006

Targeted disruption of the pregnancy-associated plasma protein-A gene is associated with diminished smooth muscle cell response to insulin-like growth factor-I and resistance to neointimal hyperplasia after vascular injury.

IGF-I is an important determinant of the vascular response to injury in large part through its ability to stimulate migration and proliferation of smooth muscle cells (SMCs). In this study, we used mice with targeted disruption of the pregnancy-associated plasma protein-A gene (PAPP-A-/-) and wild-type (WT) littermates to test the hypotheses that PAPP-A, a metalloproteinase that cleaves inhibitory IGF binding protein (IGFBP)-4, regulates vascular SMC responses to IGF-I in vitro and is critical for the development of vascular neointima after injury in vivo. Vascular SMCs from PAPP-A-/- mice lacked IGFBP-4 protease activity and failed to respond to treatment with IGF-I in the presence of IGFBP-4, whereas SMCs from WT mice with robust IGFBP-4 protease activity showed significant migratory and proliferative responses to IGF-I/IGFBP-4. For in vivo testing, PAPP-A-/- and WT mice underwent unilateral carotid ligation, a model of injury-induced neointimal hyperplasia. In WT mice, PAPP-A mRNA expression was markedly elevated 7 and 14 d after carotid ligation, associated with a progressive increase in neointimal hyperplasia and, in many cases, with complete occlusion of the vessel at 28 d. In contrast, PAPP-A-/- mice showed little evidence of progression resulting in a 75% reduction in neointimal area when compared with WT at 28 d. Cells staining for proliferating cell nuclear antigen were plentiful in the SMC-rich medial and neointimal areas of the injured WT vessel in stark contrast to the relatively few proliferating cells in the same areas of the PAPP-A-/- vessel. Expression of IGF-I and IGFBP-4 was similarly elevated in injured carotids from WT and PAPP-A-/- mice with no change in IGF-I receptor expression. IGFBP-5, an IGF-responsive gene, was increased 2-fold in WT but not in PAPP-A-/- carotids, suggesting reduced IGF activity in the absence of PAPP-A. Thus, PAPP-A-deficient mice are resistant to neointimal formation after injury, which may be explained in part by the ability of PAPP-A to enhance local IGF-I stimulation of vascular SMCs through proteolysis of IGFBP-4.

Animals↗
PubMed2006

Magnetic forces enable rapid endothelialization of synthetic vascular grafts.

BACKGROUND: Synthetic vascular grafts cannot be used in small vessels because of graft failure caused by thrombosis and neointima formation. Rapid endothelialization may overcome this limitation. We hypothesized that a magnetic graft would be able to capture and retain endothelial cells labeled with paramagnetic particles. METHODS AND RESULTS: Porcine blood derived endothelial cells were allowed to endocytose superparamagnetic iron oxide microspheres. Cell survival was assessed by trypan blue exclusion and demonstrated a dose-dependent cell survival of 75% to 95%. A flexible magnetic sheet was annealed to the external surface of a knitted Dacron graft. Labeled cells (10(6)/mL) were placed within the graft for 5 minutes. Confocal and electron microscopy confirmed uniform cell capture at the magnetized surface. The effect of shear forces on the adherent cells was evaluated in a flow chamber. The cells remained attached at rates up to 300 mL/min, with cell loss commencing at 400 mL/min. Prototype magnetic grafts were implanted in porcine carotid arteries. Labeled cells were placed within the graft for 10 minutes at the time of implantation. The grafts were evaluated after one day and uniform cell coverage was noted on the magnetized surface. In comparison, relatively few labeled cells were seen attached to a nonmagnetized surface. CONCLUSIONS: Magnetic forces can be used to rapidly cover a vascular graft with paramagnetically labeled cells. This biophysical interaction is sufficient to retain cells in the presence of blood flow. Applications of this technique may include rapid endothelialization of synthetic vascular grafts and dialysis fistulas.

Animals↗
PubMed2006

Cardiac cell-repair therapy: clinical issues.

The possibility that cardiac cell-repair therapy might become a clinical reality is a challenge worthy of the current state of technological and scientific expertise at the start of the 21(st) century. The success of preclinical and early clinical studies is a strong inducement to move ahead with larger clinical trials, but caution is warranted given our lack of understanding of the potential mechanisms by which cell-repair therapy exerts a benefit on ventricular function, perfusion, and infarct size, irrespective of the type of cell, method, site, and disease entity. There are multiple clinical, mechanistic, and safety questions requiring answers, and these will be forthcoming only if the design of clinical trials is carefully tailored to answer specific questions. These questions, in turn, will require the use of different and multiple end points, depending on the specific issue and study. Accordingly, this review addresses the limitations of current clinical studies, the design of future trials, and the concept of a hierarchical series of end points that might provide answers to a host of different questions. Clinical and basic scientists need to approach the next generation of trials in partnership.

Animals↗
PubMed2005

Gene transfer for ischemic cardiovascular disease: is this the end of the beginning or the beginning of the end?

The past decade has represented a period of high expectations for cardiovascular gene transfer on the basis of the findings from preclinical experiments and promising early clinical results. Yet, randomized studies have not demonstrated similar results. Do these poor results mean that gene transfer for ischemic cardiovascular disease has failed in its promise, or do they merely signify the inherent challenges of a pioneering field? In this paper we briefly review the clinical experience of gene transfer for ischemic cardiovascular disease and propose future directions for research.

Arterial Occlusive Diseases↗
PubMed2005

Therapeutic uses of autologous endothelial cells for vascular disease.

Endothelial cells play important structural and functional roles in vascular homoeostasis. Perturbations in endothelial cell number and function are directly involved with the initiation and progression of multiple cardiovascular diseases, including atherosclerosis, hypertension and congestive heart failure. Attempts to modify these disorders have included pharmacological strategies to improve vascular and thus endothelial function. A goal of biological approaches to these disorders is the delivery of endothelial cells that might act to provide beneficial endothelial-derived factors. However, this approach has generally been limited by the lack of readily available autologous endothelial cells for delivery. The isolation of circulation-derived endothelial progenitor cells allows for direct access to autologous endothelial cells for preclinical and clinical studies. Preclinical studies using autologous endothelial cells have demonstrated beneficial effects when delivered in animal models of vascular injury and grafting. These effects are related to the endothelial nature of the cells and may be paracrine in nature. Ongoing studies are aimed at defining the nature of these effects and optimizing delivery strategies cognizant of these mechanisms.

Animals↗
PubMed2004

Vascular endothelial growth factor (VEGF) gene transfer enhances surgical revascularization of necrotic bone.

Avascular necrosis of bone is a relatively common clinical condition caused by inflammatory conditions, steroid or other drug use, and trauma that affect many different sites in man. Revascularization of the necrotic bone is slow to occur, often resulting in bone resorption and eventual collapse of the involved bone. Rapid revascularization and subsequent bone remodeling may lead to improved outcomes. Surgical revascularization with arterovenous bundles (AV bundles) or vascularized bone grafts results in neoangiogenesis and bone remodeling. Gene transfer of an angiogenic factor to the vessel wall may be an additional strategy to further accelerate this process. In this study, we examined the effectiveness of vascular endothelial growth factor (VEGF) gene transfer to augment surgical revascularization of necrotic bone. An adenoviral vector, either with the VEGF gene (VEGF-A) or identical virus without the cDNA VEGF insert (ADV-DeltaE1) was used to transduce endothelial cells in rabbit saphenous arteries. The artery was then placed with its venae comitantes as an AV bundle into necrotic iliac crest bone in vivo. Angiogenesis in the necrotic bone was quantified by bone blood flow measurement and assessment of vessel density following microangiography. The extent of neoangiogenesis was significantly greater in the VEGF group than the control group at 1 week postoperatively.

Animals↗
PubMed2004

Genomic profiling of the human heart before and after mechanical support with a ventricular assist device reveals alterations in vascular signaling networks.

Mechanical unloading of the heart with a left ventricular assist device (LVAD) significantly decreases mortality in patients with heart failure. Moreover, it provides a human model to define the critical regulatory genes governing myocardial remodeling in response to significant reductions in wall stress. Statistical analysis of a gene expression library of 19 paired human heart samples harvested at the time of LVAD implant and again at explant revealed a set of 22 genes that were downregulated and 85 genes that were upregulated in response to mechanical unloading with a false discovery rate of less than 1%. The analysis revealed a high percentage of genes involved in the regulation of vascular networks including neuropilin-1 (a VEGF receptor), FGF9, Sprouty1, stromal-derived factor 1, and endomucin. Taken together these findings suggest that mechanical unloading alters the regulation of vascular organization and migration in the heart. In addition to vascular signaling networks, GATA-4 binding protein, a critical mediator of myocyte hypertrophy, was significantly downregulated following mechanical unloading. In summary, these findings may have important implications for defining the role of mechanical stretch and load on autocrine/paracrine signals directing vascular organization in the failing human heart and the role of GATA-4 in orchestrating reverse myocardial remodeling. This unbiased gene discovery approach in paired human heart samples has the potential to provide critical clues to the next generation of therapeutic treatments aimed at heart failure.

Adult↗
PubMed2004

Long-term retinal transgene expression with FIV versus adenoviral vectors.

PURPOSE: Gene therapy for chronic retinal diseases will require long-term expression of therapeutic transgenes. Lentiviral and adenoviral (Ad) vectors are gene delivery systems with markedly different properties. Lentiviral vectors require integration into the host genome, which facilitates long-term expression, while Ad vectors remain episomal. We compared time course, location, and extent of transgene expression from replication-deficient feline immunodeficiency virus (FIV) vectors and Ad vectors in neonatal rat retina. METHODS: A dose-response study was conducted to determine the optimal subretinal dose for comparison of FIV and Ad vectors with an internal cassette expressing beta-galactosidase under transcriptional control of the CMV immediate-early gene promoter/enhancer. Forty-two five-day old Sprague-Dawley rats received subretinal injections of 2 microl containing 2x10(3) transducing units (TU, n=14), 2x10(4) TU (n=14) or 2x10(5) TU (n=14) of FIV vector (right eye) and Ad vector (left eye). Expression was evaluated 48 h after transduction. In the subsequent long-term expression study, 60 five-day old rats received a subretinal injection of 2x10(5) TU FIV vector (right eye) and Ad vector (left eye). Ten pairs of eyes were analyzed at 1 week, 1 month, 3 months, 6 months, 12 months, and the remainder at 16 months. Eye cups were evaluated in a masked manner for extent of beta-galactosidase expression (graded 0-5) by whole mount microscopy and by cross sectional histology. RESULTS: In the dose-response study, 2x10(5) TU resulted in consistent, widespread retinal transduction with both vectors and was selected as the dose for the subsequent study. In the long-term expression study, FIV vector resulted in a higher grade of expression than Ad at multiple single time points and produced higher overall expression when data from all eyes across the entire 16 month study were analyzed (p=0.01). Retinal expression was present at 16 months with both vectors. beta-galactosidase expression was limited to the retinal pigment epithelium (RPE) until the first month, but later was also found to a lesser extent in neurosensory retina with each vector. In contrast to FIV, most Ad injected eyes showed signs of focal accumulation of macrophage-like cells with disrupted retinal architecture. CONCLUSIONS: Both FIV and Ad vectors result in long-term transgene expression in RPE after subretinal injection. FIV vectors show more promise than Ad as delivery systems for retinal diseases since they transduce greater areas of RPE, result in less cellular infiltrate, and cause less disruption of retinal architecture. The persistent expression at 16 months of follow-up suggests that these lentiviral vectors are useful for gene therapy of chronic retinal diseases.

Adenoviridae↗
PubMed2004

Modulation of the vascular response to injury by autologous blood-derived outgrowth endothelial cells.

Delivery of a heterogeneous population of cells with endothelial phenotype derived from peripheral blood has been shown to improve vascular responses after balloon arterial injury in an endothelium-dependent manner. Refinement of culture techniques has enabled the generation of outgrowth endothelial cells (OECs), a homogeneous population of distinctly endothelial cells expanded from circulating progenitor cells. The present study tested the hypothesis that OEC delivery would confer vascular protection after balloon arterial injury in a rabbit model. Rabbit peripheral blood mononuclear cells (PBMCs) were cultured in endothelial growth medium for 4-5 wk, yielding proliferative OECs with distinct endothelial phenotype (morphology, incorporation of acetylated LDL, and expression of endothelial nitric oxide synthase and caveolin-1 but not CD14). Animals underwent balloon carotid injury immediately followed by local delivery of autologous OECs for 20 min. Fluorescent-labeled OECs were detected in all layers at 4 wk, with immunostaining revealing maintenance of endothelial phenotype (von Willebrand factor-positive and RAM-11-negative) by luminal and nonluminal cells. To evaluate functional effects, additional animals received autologous OECs, saline, or freshly harvested PBMCs as noncultured cell controls by local dwell after balloon injury. Local OEC delivery improved endothelium-dependent vasoreactivity (P < 0.05 vs. saline and PBMC) and similarly reduced neointimal formation (P < 0.05 vs. saline and PBMC). These data suggest that OECs can be detected in injured arterial segments at 4 wk. Moreover, delivery of OECs confers greater vascular protection than PBMCs or saline controls and may thus offer a novel, autologous strategy to limit the response to mechanical injury.

Animals↗
PubMed2004

Gene transfer of a novel vasoactive natriuretic peptide stimulates cGMP and lowers blood pressure in mice.

Dendroaspis natriuretic peptide (DNP) is a recently described peptide produced by Dendroaspis angusticeps with structural and functional similarities to mammalian natriuretic peptides. These similarities suggest a potential role for DNP in cardiovascular therapeutics. To determine the physiological effects of chronic delivery of DNP, a gene transfer approach using first generation adenoviral vectors was utilized. Although the gene for DNP has not been cloned in any species, the peptide sequence in the snake is known. Preferred mammalian codons for snake DNP were cloned downstream of either the leader sequence (referred to as pBDNP-1) or prepropeptide sequence of human brain natriuretic peptide (BNP) cDNA (referred to as pBDNP-2). Transfections with pBDNP-1 or pBDNP-2 resulted in expected forms of chimeric DNP (cDNP) in cell lysates and conditioned media. Functional studies demonstrated the ability of both forms of cDNP within conditioned media to stimulate cGMP production in human vascular smooth muscle cells (hVSMC). Expressed cDNP inhibited hVSMC proliferation and stimulated vasorelaxation in a similar fashion. To investigate the chronic physiological effects of administration of cDNP, an adenoviral vector expressing cDNP (Ad-BDNP) was generated. Intravenous delivery of Ad-BDNP in mice resulted in dose-dependent systemic expression of cDNP. The highest level of expression was associated with consistent elevation of its presumed second messenger (cGMP) for 21 days but with transient lowering of systolic blood pressure in normotensive mice. This study demonstrates the biological features of the expression of the xenogenic peptide DNP.

Adenoviridae↗
PubMed2004

Autologous cell-based therapies for vascular disease.

Strategies that enhance the number of endothelial cells (ECs) in the vessel wall following injury may limit complications such as thrombosis, vasospasm, and neointimal formation through reconstitution of a luminal barrier and cellular secretion of paracrine factors. Proof of principle has been demonstrated by studies in which mature ECs, culture expanded from harvested vascular tissue, were seeded in the arterial wall following balloon injury. The recent identification of circulating cells capable of developing an endothelial phenotype, including progenitor cells, has raised the possibility of using blood-derived cells as therapeutic agents. This article reviews data suggesting that such cells confer vascular protective effects after injury, raising the potential for novel, autologous approaches to the treatment of vascular disease.

Animals↗
PubMed2004

Use of blood outgrowth endothelial cells as virus-producing vectors for gene delivery to tumors.

Cell-based delivery of therapeutic viruses has potential advantages over systemic viral administration, including attenuated neutralization and improved viral targeting. One of the exciting new areas of investigation is the potential ability of endothelial-lineage cells to deliver genes to the areas of neovascularization. In the present study, we compared two types of endothelial-lineage cells [outgrowth endothelial cells (OECs) and culture-modified mononuclear cells (CMMCs), also known as "endothelial progenitor cells"] for their ability to be infected with adenovirus and to home to the areas of neovascularization. Both cell types were isolated from peripheral blood of healthy human donors and expanded in culture. We demonstrate that OECs are more infectable and home better to tumors expressing VEGF on systemic administration. Furthermore, we used an adenoviral/retroviral chimeric system to convert OECs to retrovirus-producing cells. When injected systemically into tumor-bearing mice, OECs retain their ability to produce retrovirus and infect surrounding tumor cells. Our data demonstrate that OECs could be efficient carriers for viral delivery to areas of tumor neovascularization.

Adenoviridae↗
PubMed2004

The effect of vascular smooth muscle cell-targeted expression of tissue factor pathway inhibitor in a murine model of arterial thrombosis.

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that regulates the extrinsic pathway of coagulation by inhibiting the factor VIIa/tissue factor (TF) catalytic complex. TFPI is expressed by both endothelial and smooth muscle cells in the vasculature and circulates at low levels. The role of local vascular TFPI in thrombosis and the development of vascular disease is unknown. To establish an experimental animal model to directly modulate smooth muscle cell-derived TFPI on the development of arterial thrombosis, transgenic mice in which a cDNA encoding murine TFPI is expressed from the murine SM22alpha promoter were generated. Expression of transgenic mRNA was 4-fold higher than the level of endogenous TFPI mRNA in arteries from transgenic mice. In situ hybridization confirmed that expression of the transgene was limited to medial vascular smooth muscle cells. Vascular TFPI activity was increased to 2 to 3-fold in carotid homogenates. There was no difference in plasma TFPI levels or hemostatic measures (PT, aPTT and tail vein bleeding times) between these mice and their wildtype littermates. In a ferric chloride-induced model of carotid thrombosis, homozygotic transgenic mice demonstrated resistance to thrombotic occlusion compared to wildtype littermates. In transgenic mice 22% occluded within 30 minutes of application while 84% of wild type mice occluded within the same time frame (p<0.01). Heterozygotic transgenic mice had an intermediate thrombotic phenotype. Taken together, these data indicated that local VSMC-specific TFPI overexpression attenuated ferric chloride-induced thrombosis without systemic or hemostatic effects. Furthermore, this transgenic mouse model should prove useful for studying the role of TFPI in the development and progression of vascular disease.

Animals↗
PubMed2003

Diverse origin and function of cells with endothelial phenotype obtained from adult human blood.

Cells with endothelial phenotype generated from adult peripheral blood have emerging diagnostic and therapeutic potential. This study examined the lineage relationship between, and angiogenic function of, early endothelial progenitor cells (EPCs) and late outgrowth endothelial cells (OECs) in culture. Culture conditions were established to support the generation of both EPCs and OECs from the same starting population of peripheral blood mononuclear cells (PBMCs). Utilizing differences in expression of the surface endotoxin receptor CD14, it was determined that the vast majority of EPCs arose from a CD14+ subpopulation of PBMCs but OECs developed exclusively from the CD14- fraction. Human OECs, but not EPCs, expressed key regulatory proteins endothelial nitric oxide synthase (eNOS) and caveolin-1. Moreover, OECs exhibited a markedly greater capacity for capillary morphogenesis in in vitro and in vivo matrigel models, tube formation by OECs being in part dependent on eNOS function. Collectively, these data indicate lineage and functional heterogeneity in the population of circulating cells capable of assuming an endothelial phenotype and provide rationale for the investigation of new cell-therapeutic approaches to ischemic cardiovascular disease.

Antigens, Differentiation↗