Perlecan Domain-V Enhances Neurogenic Brain Repair After Stroke in Mice.

The extracellular matrix fragment perlecan domain V is neuroprotective and functionally restorative following experimental stroke. As neurogenesis is an important component of chronic post-stroke repair, and previous studies have implicated perlecan in developmental neurogenesis, we hypothesized that domain V could have a broad therapeutic window by enhancing neurogenesis after stroke. We demonstrated that domain V is chronically increased in the brains of human stroke patients, suggesting that it is present during post-stroke neurogenic periods. Furthermore, perlecan deficient mice had significantly less neuroblast precursor cells after experimental stroke. Seven-day delayed domain V administration enhanced neurogenesis and restored peri-infarct excitatory synaptic drive to neocortical layer 2/3 pyramidal neurons after experimental stroke. Domain V's effects were inhibited by blockade of α2ß1 integrin, suggesting the importance of α2ß1 integrin to neurogenesis and domain V neurogenic effects. Our results demonstrate that perlecan plays a previously unrecognized role in post-stroke neurogenesis and that delayed DV administration after experimental stroke enhances neurogenesis and improves recovery in an α2ß1 integrin-mediated fashion. We conclude that domain V is a clinically relevant neuroprotective and neuroreparative novel stroke therapy with a broad therapeutic window.

Heparan sulfate proteoglycan promotes fibroblast growth factor-2 function for ischemic heart repair.

It is well known that the basic fibroblast growth factor (bFGF) promotes angiogenesis after myocardial infarction (MI), but its biological functions decrease in the event of diffusion, enzymolysis, and weak binding with co-receptors in vivo. Heparan sulfate proteoglycans (HSPG) are a major component of extracellular matrices and have been shown to regulate a wide range of cellular functions and bioprocesses by acting as a co-receptor for bFGF and affecting its bioactivities. However, the influence of HSPG on the function of bFGF after myocardial infarction is unknown. Here, exogenous HSPG along with bFGF was injected into the hearts of rats to deliver the angiogenic growth factor for ischemic heart repair following induced MI. The specific binding of HSPG with bFGF protein was demonstrated, which was about 6-fold stronger than the binding of bFGF with heparin. The biological mechanisms of HSPG binding with bFGF were further studied by cell adhesion assay, and assays of bFGF and matrix metalloproteinase 2 (MMP2) activities demonstrated that HSPG enhances cell adhesion, promotes the bioactivity of bFGF in angiogenesis, and protects bFGF from enzymolysis. Our results indicate that HSPG has potential clinical utility as a delivery agent for heparin-binding growth factors. Additionally, HSPG shows high binding affinities with different ECM proteins which also help to anchor bFGF to heart tissue. Therefore, extracellular proteins that mimic the bio-scaffold of the extracellular matrix could promote the activities of bFGF to facilitate ischemic heart repair.

Perlecan regulates pericyte dynamics in the maintenance and repair of the blood-brain barrier.

Ischemic stroke causes blood-brain barrier (BBB) breakdown due to significant damage to the integrity of BBB components. Recent studies have highlighted the importance of pericytes in the repair process of BBB functions triggered by PDGFRß up-regulation. Here, we show that perlecan, a major heparan sulfate proteoglycan of basement membranes, aids in BBB maintenance and repair through pericyte interactions. Using a transient middle cerebral artery occlusion model, we found larger infarct volumes and more BBB leakage in conditional perlecan (Hspg2)-deficient (Hspg2 - / - -TG) mice than in control mice. Control mice showed increased numbers of pericytes in the ischemic lesion, whereas Hspg2 - / - -TG mice did not. At the mechanistic level, pericytes attached to recombinant perlecan C-terminal domain V (perlecan DV, endorepellin). Perlecan DV enhanced the PDGF-BB-induced phosphorylation of PDGFRß, SHP-2, and FAK partially through integrin α5ß1 and promoted pericyte migration. Perlecan therefore appears to regulate pericyte recruitment through the cooperative functioning of PDGFRß and integrin α5ß1 to support BBB maintenance and repair following ischemic stroke.

Extensive Transduction and Enhanced Spread of a Modified AAV2 Capsid in the Non-human Primate CNS.

The present study was designed to characterize transduction of non-human primate brain and spinal cord with a modified adeno-associated virus serotype 2, incapable of binding to the heparan sulfate proteoglycan receptor, referred to as AAV2-HBKO. AAV2-HBKO was infused into the thalamus, intracerebroventricularly or via a combination of both intracerebroventricular and thalamic delivery. Thalamic injection of this modified vector encoding GFP resulted in widespread CNS transduction that included neurons in deep cortical layers, deep cerebellar nuclei, several subcortical regions, and motor neuron transduction in the spinal cord indicative of robust bidirectional axonal transport. Intracerebroventricular delivery similarly resulted in widespread cortical transduction, with one striking distinction that oligodendrocytes within superficial layers of the cortex were the primary cell type transduced. Robust motor neuron transduction was also observed in all levels of the spinal cord. The combination of thalamic and intracerebroventricular delivery resulted in transduction of oligodendrocytes in superficial cortical layers and neurons in deeper cortical layers. Several subcortical regions were also transduced. Our data demonstrate that AAV2-HBKO is a powerful vector for the potential treatment of a wide number of neurological disorders, and highlight that delivery route can significantly impact cellular tropism and pattern of CNS transduction.

Perlecan domain IV peptide stimulates salivary gland cell assembly in vitro.

Treatment of xerostomia would benefit from development of a functional implantable artificial salivary gland. Salivary gland tissue from surgical patients was assessed by histology and immunohistochemistry to establish the phenotype of normal salivary gland cells including the native basement membranes. Ductal and acinar cells were identified in tissue and cultured cells from dispersed tissue. High levels of laminin and perlecan/HSPG2 (heparan sulfate proteoglycan 2) were noted in basement membranes, and perlecan also was secreted and organized by cultured acinar populations, which formed lobular structures that mimicked intact glands when cultured on Matrigel or a bioactive peptide derived from domain IV of perlecan. On either matrix, large acini-like lobular structures grew and formed connections between the lobes. alpha-Amylase secretion was confirmed by staining and activity assay. Biomarkers, including tight junction protein E-cadherin and water channel protein aquaporin 5 found in tissue, were expressed in cultured acinar cells. Cells cultured on Matrigel or domain IV of perlecan peptide organized stress fibers and activated focal adhesion kinase. We report a novel technique to isolate acinar cells from human salivary gland and identify a human peptide sequence in perlecan that triggers differentiation of salivary gland cells into self-assembling acini-like structures that express essential biomarkers and which secrete alpha-amylase.

Endorepellin in vivo: targeting the tumor vasculature and retarding cancer growth and metabolism.

BACKGROUND: The antiangiogenic approach to controlling cancer requires a better understanding of angiogenesis and the discovery of new compounds that modulate this key biological process. Here we investigated the role of endorepellin, an angiostatic protein fragment that is derived from the C-terminus of perlecan, a heparan sulfate proteoglycan, in controlling tumor angiogenesis in vivo. METHODS: We administered human recombinant endorepellin systemically to mice bearing orthotopic squamous carcinoma xenografts or syngeneic Lewis lung carcinoma tumors. We monitored tumor growth, angiogenesis, metabolism, hypoxia, and mitotic index by using quantitative immunohistochemistry and positron emission tomography scan imaging. In addition, we determined the localization of injected endorepellin using near-infrared labeling and immunohistochemistry of frozen tumor sections. Finally, we isolated tumor-derived endothelial cells and tested whether endorepellin could interact with these cells and disrupt in vitro capillary morphogenesis. All statistical tests were two-sided. RESULTS: Endorepellin specifically targeted the tumor vasculature as determined by immunohistochemical analysis and accumulated in the tumor perivascular zones where it persisted for several days as discrete deposits. This led to inhibition of tumor angiogenesis (as measured by decreased CD31-positive cells, mean control = 1902 CD31-positive pixels, mean endorepellin treated = 343.9, difference between means = 1558, 95% confidence interval [CI] = 1296 to 1820, P<.001), enhanced tumor hypoxia, and a statistically significant decrease in tumor metabolism and mitotic index (as measured by decreased Ki67-positive cells, mean control Ki67 pixels = 5970, mean endorepellin-treated Ki67 pixels = 3644, difference between means = 2326, 95% CI = 1904 to 2749, P<.001) compared to untreated controls. Endorepellin was actively internalized by tumor-derived endothelial cells causing a redistribution of alpha2beta1 integrin such that both proteins colocalized to punctate deposits in the perivascular region. Endorepellin treatment inhibited in vitro capillary morphogenesis of both normal and tumor-derived endothelia. CONCLUSIONS: Our results provide support for the hypothesis that endorepellin is an effective antitumor vasculature agent that could be used as a therapeutic modality to combat cancer.

The role of heparan sulfate and perlecan in bone-regenerative procedures.

Tissue engineering, grafting procedures, regeneration, and tissue remodeling are developing therapeutic modalities with great potential medical value, but these regenerative modalities are not as effective or predictable as clinicians and patients would like. Greater understanding of growth factors, cytokines, extracellular matrix molecules, and their roles in cell-mediated healing processes have made these regenerative therapies more clinically viable and will continue advancing the fields of tissue engineering and grafting. However, millions of oral and non-oral bone-grafting procedures are performed annually, and only a small percentage yield the most desirable results. Here we review the heparan-sulfate-decorated extracellular biomolecule named perlecan and the research relating to its potential as an adjunct in bone-regenerative procedures. The review includes an overview of bone graft substitutes and biological adjuncts to bone-regenerative procedures in medicine as they apply to periodontal disease, alveolar ridge augmentation, and barrier membrane therapy. Perlecan is discussed as a potential biological adjunct in terms of growth factor sequestration and delivery, and promoting cell adhesion, proliferation, differentiation, and angiogenesis. Further, we propose delivery and application schemes for perlecan and/or its domains in bone-regenerative procedures, with particular emphasis on its heparan-sulfate-decorated domain I. The perlecan molecule, with its heparan sulfate glycosylation, may provide a multi-faceted approach for the delivery of a more comprehensive stimulus than other single potential adjuncts currently available for bone-regenerative procedures.