Synthetic Fibronectin Peptides Research Articles

Synthetic fibronectin peptide exerts neuroprotective effects on transient focal brain ischemia in rats

Leukocytes have been investigated during the past decade for their roles in secondary tissue damage after ischemia/reperfusion injury. Peptide PRARIY, a synthetic fibronectin peptide, has shown an anti-adhesion effect in in vitro studies. Previous studies have demonstrated that anti-adhesion agents lead to reductions in apoptosis. The purpose of the present study was to determine whether the peptide PRARIY displays anti-inflammatory, anti-apoptotic, and neuroprotective effects following transient focal brain ischemia in rats. Twenty-six male Sprague–Dawley rats (300–350 g) were randomly divided into three groups: phosphate-buffered saline (PBS) controls, PRARI controls, and PRARIY treatments. The right middle cerebral artery was transiently occluded using a 4-0 nylon suture. One hour later, the occluder was withdrawn, and reperfusion was maintained for 48 h. Immediately after reperfusion, the peptides (20 mg/kg, dissolved in PBS) and the same volume of PBS were continuously infused through the right external carotid artery using an osmotic minipump for 24 h. Neurological deficits were examined at 3, 24, and 48 h after ischemia. Forty-eight hours after reperfusion, the rats were sacrificed for determining infarction size, leukocyte infiltration, and apoptosis in the ischemia area. Unexpectedly, PRARIY did not influence leukocyte infiltration. However, PRARIY-treated rats showed significantly functional outcome, reduction of infarction size, decrease of TUNEL positive cells, and increase of Bcl-2 (anti-apoptotic protein) positive cells in the ischemic areas when compared to the controls. These data indicate that the peptide PRARIY exerts its neuroprotective effects via supporting neural cell survival rather than anti-leukocyte recruitment following brain ischemia/reperfusion injury.

Enhancement of nigral graft survival in rat brain with the systemic administration of synthetic fibronectin peptide V

A major obstacle in neural transplantation is a severe loss of neurons in grafts soon after implantation. In the present study, we have investigated whether the systemic administration of synthetic fibronectin peptide V can increase the survival of neural grafts. Synthetic fibronectin peptide V is derived from the 33,000 mol. wt carboxyl-terminal heparin-binding domain of fibronectin. Previous studies have shown that these polypeptides possess anti-inflammatory properties. However, it is currently unknown whether this peptide has anti-apoptotic properties. Dissociated neural grafts were prepared from the ventral mesencephalon of pregnant Sprague–Dawley rats and were stereotaxically injected as a cell suspension into the striatum of adult Sprague–Dawley rats. A group of recipient rats received i.v. injections of peptide V (5 mg/kg, dissolved in saline) at 24 and 4 h prior to transplantation, at the time of transplantation, and 24, 48 and 72 h post-transplantation. Saline-treated rats served as controls. The rats were killed at two, four and 42 days post-grafting and the brain tissue was immunologically processed for tyrosine-hydroxylase, major histocompatibility complex class I and class II antigens, complement receptor type 3 and leukocyte common antigen immunocytochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. We found a significant increase (approximately twofold) in the number of dopamine neurons in the grafts for the peptide-treated group at four and 42 days compared with the controls. In contrast, there was no significant difference in the patterns of inflammation using different immunocytochemical markers in the two different groups. The levels of expression for these markers, however, were reduced over time. Interestingly, the number of apoptotic cells in the graft areas was significantly smaller in the peptide-treated group than in the control group two days after grafting. The results demonstrate that the systemic administration of synthetic fibronectin peptide V can dramatically increase the survival of nigral grafts in the brain and substantially reduce the number of apoptotic cells in the graft site, suggesting that this peptide may exert a beneficial effect on survival of nigral grafts through an anti-apoptotic mechanism.

Extracellular matrix proteins in organ transplantation.

Extracellular matrix proteins in organ transplantation.

Inhibition of leukocyte-mediated tissue destruction by synthetic fibronectin peptide (Trp-9-Tyr).

Burns are surrounded by an inflammatory zone of stasis that can progress to ischemia and extension of burn size. Synthetic fibronectin peptides have reduced tissue destruction in several models of inflammation. In this study, we postulate that administration of the peptide Trp-9-Tyr will alter the progression of tissue destruction following thermal injury. Baseline cutaneous blood flow was measured on New Zealand White rabbits with a laser doppler blood-flow meter. While the rabbits were under general anesthesia, 6 full-thickness burns were produced on the rabbits' backs. Blood flow in the zones of stasis was followed daily, and the number of zones that progressed to necrosis was determined at 72 hours. There were 3 experimental groups. Ten control animals received saline. Ten were treated with Trp-9-Tyr for 24 hours postburn. Ten received Trp-9-Tyr for 48 hours. Animals treated with Trp-9-Tyr had higher blood flow and less necrosis in the zones of stasis than did control animals, which was evident at 24 hours but more significant at 48 hours.

Abstracts of Literature

Abstracts of Literature

Antagonism of Leukocyte Adherence by Synthetic Fibronectin Peptide V in a Rat Model of Transient Focal Cerebral Ischemia

OBJECTIVE: Activated polymorphonuclear leukocytes (PMNs) seem to be directly involved in potentiating ischemic brain injury. Recent work in our laboratory demonstrated that synthetic fibronectin peptides significantly inhibit PMN accumulation in ischemic tissue, reduce the size of infarction, and reduce neurological dysfunction after transient focal cerebral ischemia in rats. The purpose of this study was to examine any dose-related effects (Experiment 1) and the optimal timing of the administration (Experiment 2) of synthetic fibronectin peptide V (FN-C/H-V) to further substantiate the role of the peptide in ameliorating cerebral ischemic damage. METHODS: Fifty-six animals were included in the study. We evaluated the efficacy of FN-C/H-V on PMN accumulation in ischemic tissue, infarct size, and neurological outcomes in rats subjected to 1 hour of cerebral ischemia and 48 hours of reperfusion. RESULTS: In Experiment 1, the animals receiving FN-C/H-V at a dose of 10 to 15 mg/kg of body weight per injection showed significant reduction of PMN accumulation, reduction of infarct size, and improvement of neurological outcomes at 48 hours after reperfusion compared to untreated animals(P < 0.05). In Experiment 2, the animals receiving FN-C/H-V within 3 hours after reperfusion also showed significantly better results than untreated animals (P < 0.05). Despite the treatment delay, the administration of FN-C/H-V inhibited PMN accumulation after reperfusion but did not reduce the size of infarction when administered 6 hours after reperfusion. CONCLUSION: These data suggest that relatively late postischemic administration of FN-C/H-V is effective in brain protection after ischemia/reperfusion.

Antagonism of leukocyte adherence by synthetic fibronectin peptide V in a rat model of transient focal cerebral ischemia.

Activated polymorphonuclear leukocytes (PMNs) seem to be directly involved in potentiating ischemic brain injury. Recent work in our laboratory demonstrated that synthetic fibronectin peptides significantly inhibit PMN accumulation in ischemic tissue, reduce the size of infarction, and reduce neurological dysfunction after transient focal cerebral ischemia in rats. The purpose of this study was to examine any dose-related effects (Experiment 1) and the optimal timing of the administration (Experiment 2) of synthetic fibronectin peptide V (FN-C/H-V) to further substantiate the role of the peptide in ameliorating cerebral ischemic damage. Fifty-six animals were included in the study. We evaluated the efficacy of FN-C/H-V on PMN accumulation in ischemic tissue, infarct size, and neurological outcomes in rats subjected to 1 hour of cerebral ischemia and 48 hours of reperfusion. In Experiment 1, the animals receiving FN-C/H-V at a dose of 10 to 15 mg/kg of body weight per injection showed significant reduction of PMN accumulation, reduction of infarct size, and improvement of neurological outcomes at 48 hours after reperfusion compared to untreated animals (P < 0.05). In Experiment 2, the animals receiving FN-C/H-V within 3 hours after reperfusion also showed significantly better results than untreated animals (P < 0.05). Despite the treatment delay, the administration of FN-C/H-V inhibited PMN accumulation after reperfusion but did not reduce the size of infarction when administered 6 hours after reperfusion. These data suggest that relatively late postischemic administration of FN-C/H-V is effective in brain protection after ischemia/reperfusion.

Antagonism of Leukocyte Adherence by Synthetic Fibronectin Peptide V in a Rat Model of Transient Focal Cerebral Ischemia

Antagonism of Leukocyte Adherence by Synthetic Fibronectin Peptide V in a Rat Model of Transient Focal Cerebral Ischemia

Neuronal protection from cerebral ischemia by synthetic fibronectin peptides to leukocyte adhesion molecules.

Leukocytes play an important role in the development of ischemia/reperfusion injury. Recent work in our laboratory has demonstrated that a mixture of synthetic fibronectin peptides to leukocyte adhesion molecules reduces ischemic brain damage after transient focal cerebral ischemia. The purpose of this study was to evaluate the efficacy of the individual peptides on leukocyte accumulation, infarct size, and neurological outcome in rats subjected to 1 h of cerebral ischemia and 48 h of reperfusion. Thirty-five animals were divided into five groups: transient ischemia without treatment (Group I), treatment with arginyl-glycyl-aspartic acid (RGD) peptide (Group II), connecting segment (CS)-1 peptide (Group III), fibronectin (FN)-C/H-V peptide (Group IV), and scrambled FN-C/H-V peptide (Group V). Groups III and IV showed a significant decrease in the degree of leukocyte infiltration in the lesion and in the infarct size (p < 0.05) when compared to Groups I, II, and V. The neurological grade of Groups III and IV was significantly better than in Groups I, II, and V at 48 h after reperfusion (p < 0.01). Thus, in addition to demonstrating the potential efficacy of synthetic peptides as therapeutic agents for ischemia-reperfusion, these results also offer new insights into the mechanisms of leukocyte arrest and recruitment in ischemia/reperfusion injury.

Autoimmune Sjögren's-like lesions in salivary glands of TGF-beta1-deficient mice are inhibited by adhesion-blocking peptides.

The targeted disruption of the TGF-beta1 gene in mice (TGF-beta1 -/-) leads to extensive inflammation in vital organs, cachexia, and death within 3 to 4 wk. Significant inflammatory lesions develop initially in the periductal regions of the salivary glands and escalate as the animals become symptomatic. These inflammatory sites, characterized by lymphocytic infiltration and increased proliferation, cytokine mRNA expression, and IgG-positive cells, resemble lesions of Sjögren's syndrome. Moreover, the inflammatory pathology, enhanced MHC expression, and Ab production are consistent with an autoimmune-like etiology. Glandular atrophy and loss of acini with reduced saliva production appear to contribute to the wasting syndrome characteristic of the TGF-beta1 -/- mice. To determine whether the structural and functional defects were developmental due to the absence of TGF-beta1 or secondary to the inflammation, TGF-beta1 -/- mice were treated with synthetic fibronectin peptides, which block leukocyte infiltration. Daily systemic injections of RGD, CS-1, and/or peptides derived from the heparin-binding region of the A chain not only prevented leukocyte infiltration in the salivary glands of the TGF-beta1 -/- mice, but also reversed the acinar and ductal derangements. These data suggested that salivary gland development is not jeopardized in the absence of TGF-beta1, but that the extensive infiltration of inflammatory cells compromises glandular structure and function. The essential nature of TGF-beta1 in controlling inflammatory and immune processes is confirmed by these studies. Moreover, these TGF-beta1 -/- mice provide an important model of autoimmune disease that can be used in the design of therapeutic interventions.

Synthetic fibronectin peptides and ischemic brain injury after transient middle cerebral artery occlusion in rats.

Leukocytes play an important role in the development of ischemia-reperfusion injury. This study was conducted to ascertain whether synthetic peptides corresponding to the cell- and heparin-binding sequences of fibronectin that disturb leukocyte adhesion molecules were effective in neuronal protection after transient focal cerebral ischemia in rats. The authors evaluated the efficacy of peptides on infarction size, leukocyte infiltration in the ischemic tissue, and neurological outcome in rats subjected to 1 hour of cerebral ischemia and 48 hours of reperfusion. Twenty-one animals were divided into three groups: transient ischemia without treatment (Group I), transient ischemia with administration of vehicle (Group II), and transient ischemia with administration of fibronectin peptides (Group III). The mean myeloperoxidase activity (U/g wet wt) in the ischemic area was as follows: Group I, 0.19% +/- 0.05; Group II, 0.21% +/- 0.03; and Group III, 0.08% +/- 0.02. The mean size of the infarction as a percentage of the total hemispheric volume was as follows: Group I, 38.35% +/- 1.34%; Group II, 39.21% +/- 2.42%; and Group III, 25.81% +/- 4.87%. Group III showed a significant decrease in myeloperoxidase activity in the lesion and the infarction size was smaller when compared to Groups I and II (p < 0.05). The neurological grade in Group III was significantly better than in Groups I and II at 48 hours after reperfusion (p < 0.01). This study is the first to explore the therapeutic potential of synthetic fibronectin peptides in brain protection after transient focal ischemia, and the results also serve as a basis for studies of important cellular and molecular events that contribute to tissue damage.

Regulation of leukocyte adhesion and signaling in inflammation and disease.

Cell adhesion molecules provide the foundation for cell communication, trafficking, and immune surveillance central to host defense. These adhesion molecules which include selectins, integrins and members of the Ig superfamily, provide a recognition system between leukocytes, endothelial cells and matrix molecules. Leukocyte-endothelial interactions initiate recruitment at sites of injury, infection and inflammation. Cell-cell and cell-matrix interactions also influence leukocyte phenotype and function. Dysregulation of these adhesion and signal transduction pathways can contribute to continued recruitment and persistent leukocyte activation with unresolved inflammation. Based on the pivotal role adhesive interactions play, the adhesion molecules provide potential targets for intervention. Selected synthetic fibronectin peptides, which inhibit leukocyte integrin binding and signal transduction in vitro, block recruitment and activation to limit inflammation in vivo.

Synthetic fibronectin peptides suppress arthritis in rats by interrupting leukocyte adhesion and recruitment.

In an experimental model of arthritis, increased leukocyte adhesion is associated with the evolution of acute and chronic synovial inflammation. Whereas peripheral blood mononuclear cells (PBMC) from control animals bind minimally to fibronectin matrices, PBMC from animals receiving arthropathic doses of bacterial cell walls demonstrate increased integrin mRNA expression and enhanced adhesion. To determine whether this augmented adhesion was causal in the development of synovial pathology, peptides synthesized from several fibronectin domains which inhibited leukocyte adhesion in vitro were administered to arthritic animals either as free peptides or coupled to a carrier molecule. Not only were peptides containing either the RGD or CS-1 cell-binding domains inhibitory to chronic synovial pathology (articular index = 10.5 +/- 0.3 for untreated animals compared to 1.25 +/- 0.25 for RGD and 2.5 +/- 0.7 for CS-1), but three peptides synthesized from the carboxy-terminal 33-kD heparin-binding domain of fibronectin were also found to significantly inhibit leukocyte recruitment and the evolution of arthritis. Based on these data, which are the first to explore the therapeutic potential of heparin-binding fibronectin peptides in chronic inflammation, it appears that antagonism of cellular adhesion and recruitment by fibronectin peptides may provide an important mechanism for modulating the multi-step adhesion process and attenuating aberrant inflammatory responses.

Synthetic fibronectin peptides interrupt inflammatory cell infiltration in transforming growth factor beta 1 knockout mice.

Pronounced mononuclear leukocyte (MNL) infiltration occurs in multiple organs of mice homozygous for a transforming growth factor beta 1 (TGF-beta 1) loss-of-function gene mutation [TGF-beta 1 (-/-)], followed by cachexia and eventually death. Consistent with the increased leukocyte adhesion and tissue infiltration, MNLs isolated from spleen, thymus, and peripheral blood of symptomatic TGF-beta 1 (-/-) mice, as compared to littermate controls, exhibited increased adhesion to extracellular matrix proteins and to endothelial cells in vitro. Incubation of TGF-beta 1 (-/-) MNLs with selected synthetic peptides corresponding to cell- and heparin-binding sequences of fibronectin (FN) significantly attenuated adhesion of these cells not only to FN but also to endothelial cells in vitro. Based on these observations, mice were treated with the FN peptides in an attempt to rescue them from tissue inflammation and cardiopulmonary failure. Daily injections of a combination of four synthetic FN peptides that interact with beta 1-integrins and/or cell surface proteoglycans blocked the massive infiltration of MNLs into the heart and lungs of TGF-beta 1 (-/-) mice. Peptide treatment initiated on day 8, coincident with the first evidence of increased leukocyte-endothelial cell interactions, not only blocked tissue infiltration but also moderated the lethal wasting syndrome.

Inhibition of tumor-cell attachment to extracellular matrix as a method for preventing tumor recurrence in a surgical wound.

Studies with four different transplantable murine tumors demonstrated that surgical instruments contaminated by contact with a tumor mass could produce tumors in a surgical wound. Eighty-seven per cent of mice with wounds made by invisibly contaminated scissors developed tumors. Irrigation with water did not prevent tumor growth. Before spilled tumor cells can invade and grow into a recurrence in the wound site, they must first attach to underlying extracellular matrix. We have devised a simple in vitro assay to identify inhibitors of tumor-cell attachment to develop therapeutic compounds that can prevent tumor-cell reimplantation. Various test compounds, including proteases (trypsin and Dispase), known modulators of matrix metabolism (proline analogues, cycloheximide, heparin, cortisone, cortexolone, and heparin-steroid combinations), large molecular weight polymers (agarose, dextran, polyethylene oxide), and synthetic fibronectin peptides were tested for their ability to inhibit mouse melanoma (B16-F10) cell attachment to gelatinized dishes. Most of these compounds had little or no effect on tumor-cell adhesion when cells were plated in serum-containing medium. However we identified three compounds that inhibited tumor-cell attachment in a reversible fashion: (1) a specific inhibitor of collagen deposition (L-azetidine-2-carboxylic acid); (2) a bacterial neutral protease (Dispase); and (3) synthetic fibronectin peptides that contained the arginine-glycine-asparate (RGD) sequence that is responsible for cell binding. Dispase and the RGD-containing peptides also inhibited cell implantation and prevented tumor formation in a surgical wound. We propose that inhibitors of attachment might be used either alone or with other biologic modifiers to prohibit implantation of free tumor cells at the time of surgery and thus, to prevent local tumor recurrence.