RESUMO
Pathological vascular smooth muscle cell (VSMC) behavior after vascular interventions such as angioplasty or bypass is initiated within the 3D environment of the vessel media. Here VSMCs proliferate, invade the surrounding matrix, migrate adluminally, and deposit substantial amounts of matrix, leading to myointimal hyperplasia and decreased blood flow to critical organs and tissue. Since focal adhesion kinase (FAK) mediates many of the VSMC responses to these pathologic events, it provides a reasonable pharmacologic target to limit this invasive VSMC behavior and to better understand the cellular pathophysiology of this disease. Here we quantified the effectiveness of disabling FAK in VSMCs with its dominant-negative inhibitor, FAK-related nonkinase (FRNK), in a clinically relevant 3D assay. We found that FRNK overexpression decreased VSMC invasion (both the length and frequency) in this matrix. These effects were demonstrated in the presence and absence of chemical mitotic inhibition, suggesting that FAK's effect on cellular matrix invasion, migration, and proliferation utilize separate and/or redundant signaling cascades. Mechanistically, FAK inhibition decreased its localization to focal adhesions which led to a significant decrease in FAK autophosphorylation and the phosphorylation of the serine/threonine kinase, AKT. Together these findings suggest that disruption of FAK signaling may provide a pharmaceutical tool that limits pathological VSMC cell behavior.
Assuntos
Fibrina/química , Regulação da Expressão Gênica/fisiologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Proteínas Tirosina Quinases/metabolismo , Actinas/metabolismo , Animais , Artérias Carótidas/citologia , Técnicas de Cultura de Células , Técnicas de Cocultura , Meios de Cultura , Cães , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Miócitos de Músculo Liso/citologia , Fosforilação , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Atherosclerotic cardiovascular disease is a leading cause of death worldwide. Despite recent improvements in medical, operative, and endovascular treatments, the number of interventions performed annually continues to increase. Unfortunately, the durability of these interventions is limited acutely by thrombotic complications and later by myointimal hyperplasia followed by progression of atherosclerotic disease over time. Despite improving medical management of patients with atherosclerotic disease, these complications appear to be persisting. Cardiovascular gene therapy has the potential to make significant clinical inroads to limit these complications. This article will review the technical aspects of cardiovascular gene therapy; its application for promoting a functional endothelium, smooth muscle cell growth inhibition, therapeutic angiogenesis, tissue engineered vascular conduits, and discuss the current status of various applicable clinical trials.
Assuntos
Doença da Artéria Coronariana/terapia , Terapia Genética/métodos , Animais , Ensaios Clínicos como Assunto , Doença da Artéria Coronariana/genética , Técnicas de Transferência de Genes , Terapia Genética/tendências , Humanos , Neovascularização Fisiológica/genética , Resultado do TratamentoRESUMO
OBJECTIVE: Cardiovascular tissue engineering approaches to vessel wall restoration have focused on the potent but relatively nonspecific and heparin-dependent mesenchymal cell mitogen fibroblast growth factor 1 (FGF-1). We hypothesized that linking FGF-1 to a sequence likely to bind to cell surface receptors relatively more abundant on endothelial cells (ECs) might induce a relative greater EC bioavailability of the FGF-1. We constructed a heparin-binding growth-associated molecule (HB-GAM)/FGF-1 chimera by linking full-length human HB-GAM to the amino-terminus of human FGF-1beta (21-154) and tested its activities on smooth muscle cells (SMCs) and ECs. METHODS: Primary canine carotid SMCs and jugular vein ECs were plated in 96-well plates in media containing 10% fetal bovine serum and grown to approximately 80% confluence. After being growth arrested in serum-free media for 24 hours, the cells were exposed to concentration ranges of cytokines and heparin, and proliferation was measured with tritiated-thymidine incorporation. Twenty percent fetal bovine serum was used as positive control, and phosphate-buffered saline was used as negative control. RESULTS: In the presence of heparin the HB-GAM/FGF-1 chimera stimulated less SMC proliferation than did the wild-type FGF-1 with a median effective dose of approximately 0.3 nmol versus approximately 0.1 nmol (P <.001). By contrast, the chimera retained full stimulating activity on EC proliferation with a median effective dose of 0.06 nmol for both cytokines. Unlike the wild-type protein, the chimera possessed heparin-independent activity. In the absence of heparin, the chimera induced dose-dependent EC and SMC proliferation at 0.06 nmol or more compared with the wild-type FGF-1, which stimulated minimal DNA synthesis at 6.0-nmol concentrations. CONCLUSIONS: The HB-GAM/FGF-1 chimera displays significantly greater and uniquely heparin-independent mitogenic activity for both cell types, and in the presence of heparin it displays a significantly greater EC specificity.
Assuntos
Proteínas de Transporte/genética , Divisão Celular/genética , Técnicas de Cultura , Citocinas/genética , Endotélio Vascular/citologia , Fator 2 de Crescimento de Fibroblastos/genética , Mitógenos , Músculo Liso Vascular/citologia , Proteínas Recombinantes de Fusão/genética , Animais , Meios de Cultura , Replicação do DNA/genética , Cães , Relação Dose-Resposta a Droga , Fator 1 de Crescimento de FibroblastosRESUMO
We have taken the short stay approach to carotid artery surgery to our VA setting over the past 5 to 6 years. Retrospectively, we reviewed the efficacy and safety of that approach in 201 consecutive carotid operations over the recent 4-year period (January 1, 1996-December 31, 1999). In 1996 we had already begun the transition to an algorithm to (1) utilize carotid color flow Doppler duplex exams for diagnosis, (2) same-day admission (SDA), (3) intensive care unit (ICU) only when deemed medically necessary, and (4) next-day discharge. Results of this approach have been a decrease in the utilization of diagnostic arteriograms and utilization of the ICU from 100% previous to the onset of this approach to 17 and 22%, respectively. SDA increased from 24 to 89%. Mean LOS decreased from 5.13+/-0.9 to 1.97+/-0.4 days. The percentage of patients completing the algorithm went from 15 to 72%. Stroke and/or death varied from 0 to 3.7% each year and was only 2.4% over the 4-year period. In conclusion, this approach to short stay carotid surgery in the veteran population has proven both efficacious and safe with results similar to those in university and community practices.
Assuntos
Artérias Carótidas/cirurgia , Tempo de Internação , Veteranos , Idoso , Feminino , Humanos , Unidades de Terapia Intensiva , Masculino , Estudos RetrospectivosRESUMO
BACKGROUND: The structure/function relationships of fibroblast growth factor 1 (FGF-1) are being investigated using site mutation, yielding novel structures with potential clinical applicability for modulating tissue responses to vascular interventions. We generated a mutant FGF-1 in which all three cysteines were converted to serines and then tested the relative mitogenic activities on endothelial cells (ECs) and smooth muscle cells (SMCs) and the molecular stability of the protein to thrombin-induced degradation. METHODS: The dose responses of wild-type FGF-1 and the Cys-free mutant in the absence or presence of heparin were tested on ECs and SMCs. Cell proliferation was measured by [(3)H]thymidine incorporation. Data were normalized by positive control (20% fetal bovine serum) and expressed as percentage of positive control for comparison. The molecular stability was examined by exposure of the cytokines to thrombin at 37 degrees C for 0.5-24 h and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. RESULTS: Unlike wild-type FGF-1 which induced only minimal DNA synthesis at concentrations as high as 100 ng/ml, the Cys-free mutant induced a dose-dependent proliferation starting at 1 ng/ml on both ECs and SMCs in the absence of heparin. At 100 ng/ml, Cys-free mutant induced 4-fold more proliferation than wild-type FGF-1 on ECs (76.64 +/- 13.39% vs 14.58 +/- 1.38%, P < 0.01) and 12-fold more proliferation on SMCs (143.52 +/- 9.96 vs 11.25 +/- 3.32, P < 0.01). Heparin 5 U/ml potentiated the mitogenic activity of the Cys-free mutant at low dose range. Both proteins were degraded by thrombin progressively. But the Cys-free mutant showed more susceptibility with accelerated appearance of lower-molecular-weight fragment bands after incubation with thrombin. CONCLUSIONS: Conversion of cysteine residues to serine changed the heparin dependency of the growth factor and increased its mitogenic activity and its susceptibility to thrombin-induced degradation.
Assuntos
Divisão Celular/efeitos dos fármacos , Cisteína , Endotélio Vascular/citologia , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/farmacologia , Heparina/farmacologia , Músculo Liso Vascular/citologia , Animais , Artérias Carótidas , Bovinos , Células Cultivadas , Cães , Endotélio Vascular/efeitos dos fármacos , Fator 1 de Crescimento de Fibroblastos , Fator 2 de Crescimento de Fibroblastos/genética , Veias Jugulares , Músculo Liso Vascular/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Soroalbumina Bovina , Relação Estrutura-AtividadeRESUMO
PURPOSE: Fibrin glue (FG) has been used for local cytokine delivery on both vascular grafts and angioplasty sites. We measured the diffusive release of vascular endothelial growth factor (VEGF) and heparin from FG and the mitogenic activity of VEGF with and without heparin in FG on canine endothelial cells (ECs) and smooth muscle cells (SMCs). METHODS: Release of VEGF labeled with iodine 125 and tritiated heparin from FG into the overlying media was serially measured over 96 hours, and the data are reported as the mean percent released +/- SD. Proliferation assays measuring tritiated thymidine incorporation were performed for ECs and SMCs plated in media with 10% serum on FG containing various concentrations of VEGF and heparin. Media was placed on the FG for 24 hours and removed before plating cells to minimize the effect of the released, soluble VEGF and heparin. RESULTS: At 24 hours, 54% +/- 1% and 58% +/- 1% of the radioactive VEGF and heparin were released, respectively, with minimal release thereafter (58% +/- 1% and 66% +/- 1% at 96 hours). The ECs, SMCs, or media only (no cells) was plated on FG containing radioactive VEGF in an immediate or 24-hour delayed fashion for 72 hours to determine the percent release of VEGF into the media with the two different methods of plating. Cell type and the presence or absence of cells did not affect VEGF release, but there was three times more VEGF in the media for the immediate versus delayed plating (P <.001). Without heparin, VEGF at 100 ng/mL or more in the FG was needed to induce EC proliferation. Heparin at 5 U/mL enhanced EC proliferation at the VEGF dose of 100 ng/mL as compared wtih no heparin (P <.001), but not at the VEGF dose of 1000 ng/mL, which likely represents a maximal response. With heparin at 500 U/mL, the ECs died. In contrast, VEGF, in the presence or absence of heparin, did not affect SMC proliferation. CONCLUSIONS: We conclude that FG with VEGF at 1000 ng/mL and heparin at 5 U/mL is the optimal concentration for in vivo use because this may encourage EC, but not SMC, proliferation. The VEGF at 1000 ng/mL should leave mitogenic concentrations of VEGF intact after the initial, diffusive loss, and the addition of heparin at 5 U/mL may enhance VEGF mitogenic activity.
Assuntos
Fatores de Crescimento Endotelial/farmacologia , Adesivo Tecidual de Fibrina , Heparina/farmacologia , Linfocinas/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Cães , Endotélio Vascular/citologia , Músculo Liso Vascular/citologia , Isoformas de Proteínas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
OBJECTIVE: Site-directed mutagenesis is an important technique that can alter cytokine function, thereby eliciting desired responses. S130K is a mutation of fibroblast growth factor-1 (FGF-1), with lysine replacing serine in the heparin-binding site. We measured molecular stability and mitogenic activity of FGF-1 and S130K, both in the media and when suspended in fibrin glue (FG), on smooth muscle cells (SMCs) and endothelial cells (ECs) to determine if the mutation altered the function and potential clinical applicability. METHODS: EC and SMC proliferation of soluble FGF-1 or S130K at 0, 0. 1, 1, 10, or 100 ng/mL with heparin at 0, 5, 50, or 500 units (U)/mL was measured on growth-arrested cells in serum-free media. EC and SMC proliferation assays with cells on FG containing either FGF-1 or S130K at 0, 1, 10, 100, or 1000 ng/mL in combination with heparin at 0, 5, 50 or 500 U/mL were also performed during the exponential growth phase. Molecular degradation by thrombin was measured by sodium dodecylsulfate-polyacrylamide gel electrophoresis. RESULTS: S130K induces greater EC and SMC proliferation in the absence of heparin than FGF-1 does (P <.0001 for both the 10 and 100 ng/mL doses). S130K is also significantly more potent than FGF-1 in the presence of heparin. Heparin in the media enhances cytokine-induced SMC and EC proliferation at doses of 5 U/mL, but inhibits SMC proliferation at concentrations of 500 U/mL. For the FG data, unlike FGF-1, S130K induces EC and SMC proliferation in the absence of heparin. The addition of 5 U/mL of heparin enhances the proliferation induced by S130K. For ECs, as the heparin dose increases to 50 U/mL, proliferation decreases, as compared with the 5 U/mL concentration when either FGF-1 or S130K in the FG was compared at concentrations of 10, 100, and 1000 ng/mL (P <.01). S130K is more potent in FG than is FGF-1 both with and without heparin and exhibits maximal EC and SMC proliferation at 10 ng/mL, whereas FGF-1 activity is maximal at 100 ng/mL. Gel electrophoresis demonstrated that S130K was relatively more resistant to thrombin degradation than FGF-1. CONCLUSIONS: Site-directed mutagenesis changed the potency and the heparin dependency on cellular proliferation of FGF-1 in vitro. These techniques should allow the delivery of mutant growth factors to areas of vascular intervention to induce specific, desired responses. We believe that these studies will enhance our knowledge of the function of various regions of the FGF-1 molecule, allowing us to more precisely design increasingly more useful FGF-1 mutants.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Adesivo Tecidual de Fibrina/farmacologia , Heparina/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Trombina/efeitos dos fármacos , Adesivos Teciduais/farmacologia , Animais , Sequência de Bases , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura Livres de Soro , Primers do DNA , Cães , Relação Dose-Resposta a Droga , Interações Medicamentosas , Endotélio Vascular/citologia , Fator 1 de Crescimento de Fibroblastos/farmacologia , Dados de Sequência Molecular , Músculo Liso Vascular/citologia , Trombina/metabolismoRESUMO
Both cardiovascular implants and therapeutic interventions on native arteries fail due to biologic responses occurring at the blood/prosthesis/arterial wall and tissue/prosthesis/arterial wall interfaces, resulting in the failure modes of thrombosis and myointimal hyperplasia. Systemic pharmacologic approaches including use of anti-coagulant and anti-platelet agents have significant untoward side effects and have not resulted in a dramatic impact on failure modes in many applications, including small diameter vascular grafts. Local delivery of therapeutic agents via surface attachment with defined release kinetics may alter thrombogenicity and/or myointimal hyperplasia. Therapeutic agents may include a spectrum of biologic agents from peptides to endothelial cells. Efficient attachment and release of these agents in biologically active form is dependent upon improved methods of surface modification. The intended action of the biologic agent may similarly be impacted by the surface and bulk characteristics of the underlying biomaterial. It is often assumed, without concrete data. that surface re-endothelialization may have a beneficial impact on both thrombogenicity and myointimal hyperplasia. New clinical data on endothelial cell seeding has been supportive. Spontaneous re-endothelialization may be stimulated via an induced directed angiogenesis resulting in trans-interstitial capillarization and surface endothelialization. Recent advances in therapeutic angiogenesis have suggested the power of angiogenic factors to induce neovascularization of ischemic tissue beds. These concepts have been used to surface modify prosthetic devices with either VEGF or FGF and both in vitro and animal data suggest a potent stimulation of surface re-endothelialization. Neither of these growth factors is likely to be ideal. VEGF is relatively endothelial cell specific but is a relatively weak endothelial cell mitogen. FGF-1 and FGF-2 are more potent mitogens but are less cell specific. Recent work has led to the generation of mutant growth factors via site-induced mutagenesis and results of several such FGF mutants on endothelial cell and smooth muscle cell proliferative response have been studied. The use of 'designer growth factors' on cardiovascular implants and on manipulated native vessels may have a significant positive impact on re-endothelialization and thereby on the failure modes of thrombosis and myointimal hyperplasia.
Assuntos
Prótese Vascular , Circulação Coronária , Endotélio Vascular/citologia , Neovascularização Fisiológica , Animais , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Substâncias de Crescimento/farmacologia , Humanos , Neovascularização Fisiológica/efeitos dos fármacosRESUMO
BACKGROUND: The fluorescent marker PKH26 has been demonstrated to be useful for the tracking of endothelial cells in short-term studies; however, the optimal labeling conditions for long-term implants have not been determined. This study was designed to evaluate the effects of PKH26 on endothelial cell proliferation and to identify labeling conditions that would yield the greatest fluorescence over time without adversely affecting cell viability. MATERIALS AND METHODS: Canine jugular vein endothelial cells (CJVECs) were labeled with 0. 04 microM PKH26. Proliferation of labeled and control cells was assessed for 8 consecutive days by [(3)H]thymidine uptake. In a second experiment, CJVECs were labeled at concentrations of 0, 5, 8, 10, and 20 micromol/L. Cells were maintained in culture for 60 days. The fluorescence intensity of each cell population was measured using two techniques. At baseline and at 60 days, fluorescence was measured using a fluorescence-activated cell sorter. On days 14, 28, 45, and 60 fluorescence was measured by constructing gray-scale histograms from photomicrographs taken of each flask under rhodamine illumination. Mean viable cell number for each concentration was determined after 60 days. RESULTS: In the first experiment, PKH26-labeled and unlabeled CJVECs demonstrated nearly identical growth curves, suggesting that PKH26 had no adverse effect on proliferation. In the second experiment, after 60 days, the 10 and 20 microM groups displayed greater fluorescence by histogram than the 0, 5, or 8 microM groups; however, they were not significantly different from each other (mean intensity 8.2 vs 9.1, P > 0.05, Student-Newman-Keuls test for multiple comparisons). Over 60 days, the cells labeled with 20 microM PKH26 experienced the only significant decrease in viable cells compared to the unlabeled group (5.5 x 10(5) vs 9.6 x 10(5) cells/flask, P < 0.05). Importantly, we observed no significant differences in cell number between the 10 microM group and the lower concentrations compared to the unlabeled cells (P > 0.05). CONCLUSIONS: We conclude that a concentration of 10 microM PKH26 provides the optimal labeling condition for endothelial cells when long-term tracking is desired.
Assuntos
Endotélio Vascular/citologia , Corantes Fluorescentes , Compostos Orgânicos , Animais , Contagem de Células/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transplante de Células , Cães , Relação Dose-Resposta a Droga , Corantes Fluorescentes/farmacologia , Técnicas In Vitro , Concentração Osmolar , Fatores de Tempo , Transplante AutólogoRESUMO
PURPOSE: The results of intraoperative and early postoperative carotid color-flow duplex scanning (CFS) after endarterectomy were reviewed to determine whether any perioperative studies could be eliminated. METHODS: Patients undergoing carotid endarterectomy with intraoperative CFS between 1986 and 1997 were identified. Early postoperative CFS was performed between 1 day and 3 weeks postoperatively, then it was performed again at 6 months postoperatively. RESULTS: During the study period, 560 patients, 325 men and 235 women, underwent 621 carotid endarterectomies. A satisfactory intraoperative carotid CFS was completed in 611 (98.4%) patients. There were 20 (3.2%) vessels with a major defect that required revision for fronds or flaps (n = 11), retained atheroma (n = 5), low flow (n = 2), high velocity or turbulence (n = 1), or dissection (n = 1). Another 146 vessels (23.5%) had minor defects, such as retained proximal atheromas or small (less than 3 mm) fronds, but were not revised. The remaining 445 vessels were normal. The first postoperative CFS was normal in all the revised carotids and in 138 (94.5%) vessels with minor intraoperative defects. At 6 months, recurrent stenosis (more than 75% area reduction) was identified in 1 of 18 revised carotids (5.5%), 4 of 138 vessels (2. 9%) with minor defects, and 17 of 406 vessels (4.2%) that were normal intraoperatively. The incidence of recurrent stenosis was not significantly different in the three groups (P =.7). CONCLUSION: Intraoperative CFS is useful because major unsuspected defects can be corrected immediately, thus avoiding potential neurologic morbidity. However, the postoperative day 1 CFS can be eliminated in most cases, because it does not provide any relevant clinical information.
Assuntos
Artérias Carótidas/diagnóstico por imagem , Estenose das Carótidas/diagnóstico por imagem , Ultrassonografia Doppler em Cores , Idoso , Estenose das Carótidas/cirurgia , Endarterectomia das Carótidas , Feminino , Humanos , Cuidados Intraoperatórios , Masculino , Pessoa de Meia-Idade , Período Pós-Operatório , Estudos RetrospectivosRESUMO
PURPOSE: Fibrin glue (FG) has been used as a delivery system for bioactive agents on grafts and angioplasty sites. Reports from two different institutions suggest that heparin concentrations of 500 U/mL in FG inhibit smooth muscle cell (SMC) proliferation, but do not effect endothelial cell (EC) proliferation. The purposes of this study were to (1) quantify the diffusive release of fibroblast growth factor-1 (FGF-1) and heparin from FG; (2) determine the effect of heparin and FGF-1 on SMC proliferation when the cells are immediately plated on the FG; and (3) by means of the diffusive release data, design a new in vitro model that may differentiate the effect of FG-incorporated FGF-1 and heparin, rather than the released, solubilized components of these two factors, on SMC and EC proliferation. METHODS: 125I-FGF-1 or 3H-heparin release from FG into the overlying media was measured serially in a 96-hour period, either with or without cells. SMCs were immediately plated on FG containing various concentrations of FGF-1 and heparin. SMCs or ECs were plated on identical groups of FG containing FGF-1 and heparin 24 hours after the FG was made to exclude the effect on cell growth of the initial release of FGF-1 into the media. RESULTS: In the first 24 hours, 70% +/- 1% of the FGF-1 and 59% +/- 2% of the heparin in the FG was released into the overlying media, with minimal release occurring thereafter. The cell type or absence of cells did not affect release, but there was five times more FGF-1 and four times more heparin in the media at 72 hours for the immediate plating versus the delayed plating because of a diffusive release primarily in the first 24 hours. A heparin concentration of 500 U/mL inhibited SMC proliferation, as compared with 5 U/mL heparin, only when immediate plating of SMCs was used. Comparing immediate versus delayed SMC plating, at equivalent FGF-1 and heparin doses, immediate plating induced greater proliferation than delayed plating; this was likely caused by the higher soluble FGF-1 concentration. Heparin doses as high as 500 U/mL had little effect on SMC proliferation. In contrast, ECs died with delayed plating on FG containing 500 U/mL heparin, and their growth was inhibited at 50 U/mL heparin, as compared with 5 U/mL heparin. CONCLUSION: The differences in SMC proliferation when comparing immediate versus delayed plating are likely caused by diffusive release of heparin and FGF-1 into the media. Our ongoing work uses an optimized in vitro FG system that minimizes the effects of soluble factors. This is an important distinction, because the cytokines that are released in vivo will be removed by blood flow and, thus, may not exert an effect unless they are contained within the FG.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Adesivo Tecidual de Fibrina , Fatores de Crescimento de Fibroblastos/farmacologia , Heparina/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Adesivos Teciduais , Animais , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura , Cães , Endotélio Vascular/citologia , Fatores de Crescimento de Fibroblastos/administração & dosagem , Heparina/administração & dosagem , Técnicas In Vitro , Músculo Liso Vascular/citologia , Suspensões , Fatores de TempoRESUMO
Wound healing is a very complex and dynamic process that involves both stimulation and inhibition of cells and bioactive substances, resulting in a stable scar. Although the histological aspects of this response are well understood, the regulation of wound healing on a molecular level has not been completely elucidated. An "appropriate" healing response is crucial after vascular graft implantation to permit a functioning patent conduit. If the biological response is unfavorable, graft failure ensues. Clearly, it has been shown that the patency for prosthetic vascular implants is less favorable than for autologous implants in certain anatomic positions. However, the factors that promote this failure have not been fully identified. This article reviews the normal biological response to vascular graft healing as we understand it and provides some alternatives to manipulate the cellular milieu in an attempt to promote a more favorable healing response to prosthetic implantation.
Assuntos
Implante de Prótese Vascular , Cicatrização , Adsorção , Animais , Proteínas Sanguíneas/metabolismo , Adesão Celular , Sobrevivência de Enxerto , Substâncias de Crescimento/fisiologia , Humanos , Propriedades de SuperfícieRESUMO
PURPOSE: The incidence rate of disease progression and stroke after the diagnosis of a moderate (50% to 79%) carotid stenosis was determined by means of color-flow duplex scanning. METHODS: During a 4-year period, 344 male veterans with moderate internal carotid artery stenoses, on one or both sides, were examined at regular intervals for a mean period of 25 months. Carotid color-flow scans were obtained semiannually. Clinical follow-up was performed to determine the incidence rate of amaurosis fugax, transient ischemic attacks, nonhemispheric symptoms, and strokes. RESULTS: New neurologic symptoms developed in 75 patients (21.8%). Fifty-one (14.8%) had ipsilateral symptoms during follow-up: 18 amaurosis fugax (5.2%), 14 transient ischemic attacks (4%), 5 nonhemispheric symptoms (1.4%), and 14 strokes (4%). Twenty-four patients (6.9%) had contralateral symptoms: 20 strokes (5.8%) and 4 transient ischemic attacks (1.2%). Life-table analysis showed that the annual rate of ipsilateral neurologic events was 8.1%, and the annual rate of stroke was 2.1%. Seventy-five patients (22%) died in the follow-up period. Disease progression to 80% to 99% stenosis or occlusion occurred in 71 of 458 vessels (15.5%). The internal carotid arteries that showed evidence of disease progression had a significantly higher initial peak systolic velocity (251 vs 190 cm/s; P <.0001) and end diastolic velocity (74 vs 52 cm/s; P < 0.0001). Black patients and patients with ischemic heart disease were at a higher risk for disease progression. We could not identify any atherosclerotic risk factors that reliably predicted patients in whom future ipsilateral neurologic symptoms were more likely to develop. However, there was an increased risk of stroke associated with progression of disease. CONCLUSION: Patients who are asymptomatic and who have moderate carotid stenoses are at significant risk for neurologic symptoms and death, but have a relatively low incidence rate of ipsilateral events. The initial flow characteristics in the stenotic vessel are predictive of future disease progression, but they are not helpful in identifying patients in whom symptoms will develop.
Assuntos
Estenose das Carótidas/complicações , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Cegueira/etiologia , Velocidade do Fluxo Sanguíneo , Artéria Carótida Interna/diagnóstico por imagem , Estenose das Carótidas/diagnóstico por imagem , Estenose das Carótidas/mortalidade , Transtornos Cerebrovasculares/etiologia , Progressão da Doença , Cobaias , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Fatores de Risco , Taxa de Sobrevida , Ultrassonografia Doppler em Cores , Ultrassonografia Doppler DuplaRESUMO
Recombinant FGF-2-SAP is a mitotoxin consisting of the plant-derived ribosome-inactivating toxin saporin (SAP) fused to basic fibroblast growth factor (FGF-2). FGF-2-SAP targets and kills cells bearing upregulated FGF receptors. In vivo, FGF-2-SAP inhibits smooth muscle cell hyperplasia in models of restenosis. The present study examined the potential for a differential effect of FGF-2-SAP on canine vascular endothelial cells (EC) and smooth muscle cells (SMC) separately as well as in a novel co-culture model. Canine vascular SMC and EC cultures were established separately and made quiescent once cells reached 80% confluence. Following the release from growth arrest, both cell types were treated with FGF-2-SAP, or FGF-2, or SAP alone for 48 h. [3H]TdR incorporation was used to determine the growth response of SMC and EC. The co-culture system was created by plating canine vascular SMC and EC on either side of a microporous 13 microm thick polyester membrane insert. Both cell types were grown to 80% confluence and independently made quiescent. Following the release from growth arrest, cells were treated with FGF-2-SAP, or FGF-2, or SAP alone. Negative and positive control groups were untreated wells containing phosphate buffered saline and complete growth media, respectively. After 48 h, both [3H]TdR incorporation and total DNA content, by fluorometric measurement, were quantitated in SMC and EC independently. FGF-2-SAP showed a concentration-dependent cytotoxicity in both canine SMC and EC but cytotoxicity for EC required substantially higher concentrations. In co-cultured SMC, FGF-2-SAP significantly decreased both [3H]TdR uptake and total DNA content at 0.5, 5, 50, and 500 ng/ml (0.01-10 nM) compared to positive controls. In co-cultured EC, FGF-2-SAP decreased [3H]TdR uptake at 50 and 500 ng/ml and total DNA content at 500 ng/ml compared to positive controls. Neither SAP alone nor FGF-2 alone showed a significant effect on [3H]TdR uptake or DNA content of either SMC or EC. In this unique co-culture model, which better replicates the relationship between SMC and EC in vivo, we demonstrated a dose-response range of FGF-2-SAP at which both the proliferation and total cell number of SMC, but not EC, is significantly reduced. These data suggest that FGF-2-SAP may have therapeutic utility in inhibiting myointimal hyperplasia in the absence of a deleterious effect on regenerating endothelium following vascular reconstructions.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/toxicidade , Imunotoxinas/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , N-Glicosil Hidrolases , Proteínas de Plantas/toxicidade , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , DNA/biossíntese , Cães , Endotélio Vascular/citologia , Hiperplasia , Músculo Liso Vascular/citologia , Proteínas Recombinantes de Fusão/toxicidade , Proteínas Inativadoras de Ribossomos Tipo 1 , Saporinas , Espectrometria de Fluorescência , TimidinaAssuntos
Procedimentos Cirúrgicos Vasculares/tendências , Centros Médicos Acadêmicos/economia , Centros Médicos Acadêmicos/tendências , Docentes de Medicina , Cirurgia Geral/educação , Humanos , Programas de Assistência Gerenciada/economia , Programas de Assistência Gerenciada/tendências , National Institutes of Health (U.S.) , Assistência ao Paciente , Pesquisa/tendências , Apoio à Pesquisa como Assunto/classificação , Apoio à Pesquisa como Assunto/economia , Apoio à Pesquisa como Assunto/tendências , Sociedades Médicas , Desenvolvimento de Pessoal/tendências , Apoio ao Desenvolvimento de Recursos Humanos/classificação , Apoio ao Desenvolvimento de Recursos Humanos/economia , Apoio ao Desenvolvimento de Recursos Humanos/tendências , Estados Unidos , Procedimentos Cirúrgicos Vasculares/economiaRESUMO
Fibrin sealant (FS) is a mixture of concentrated fibrinogen and thrombin that creates a fibrin matrix that is slowly degraded by the body's fibrinolytic system. FS is currently being used in the clinical arena for many applications. Perhaps the most relevant indication for vascular surgeons concentrates on FS's hemostatic properties. Current research in many centers is investigating FS's capability to incorporate drugs and cytokines into the fibrin matrix for slow release as a drug delivery system for future clinical use. This review will focus on three main uses of FS: as an anastomotic sealant, as an antibiotic coating, and as an agent for endothelialization of grafts.
Assuntos
Adesivo Tecidual de Fibrina , Adesivos Teciduais , Procedimentos Cirúrgicos Vasculares , Animais , Antibacterianos/administração & dosagem , Prótese Vascular , Endotélio Vascular/ultraestrutura , Hemostáticos , Humanos , Infecções Relacionadas à Prótese/prevenção & controleRESUMO
BACKGROUND: The early success rates of endarterectomy and angioplasty are influenced by the thrombogenicity of the deendothelialized surface. We previously reported decreased platelet deposition after 30 and 120 minutes and after 28 days on expanded polytetrafluoroethylene (ePTFE) grafts coated with fibrin glue (FG) containing fibroblast growth factor type 1 (FGF-1) and heparin in canine aortoiliac bypass grafts when compared with control uncoated grafts. The FG/FGF-1/heparin coating has been shown to enhance spontaneous endothelialization at 28 days in canine ePTFE bypass grafts. The current study evaluates the thrombogenicity of this FG/FGF-1/heparin suspension applied to a balloon de-endothelialization model of endarterectomy in canine carotid arteries. METHODS: Nine dogs underwent bilateral, deendothelialization balloon injury to 6-cm segments of their carotid arteries. Fibrin glue (fibrinogen 32.1 mg/mL + thrombin 0.32 U/mL) containing FGF-1 (11 ng/mL) and heparin (250 U/mL) was applied to the luminal surface of one carotid artery in each dog. Both femoral arteries were circumferentially dissected but not balloon injured; one femoral artery was clamped for the same period as the carotid arteries. In the 6 acute dogs, 10 minutes prior to the restitution of flow in both carotid arteries and one femoral artery, 4 to 8 x 10(9) (111)In-labelled autologous platelets were injected intravenously. Four-cm segments of both carotid and femoral arteries were excised after 15 or 120 minutes of circulation (n = 3/time/artery, 24 arteries). In the 3 chronic dogs, the radiolabelled platelets were injected 30 days after carotid injury. The carotid and femoral vessels were then excised after 120 minutes of perfusion. Radioactive platelet deposition was quantitated by gamma counting. RESULTS: After 2 hours, the injured carotid arteries demonstrated significantly more platelet deposition than either uninjured femoral artery controls (P < 0.001). There was also a significant 45.2% decrease (P = 0.008) in platelet deposition on the balloon injured carotid arteries treated with FG/FGF-1/heparin when compared with balloon injured carotid arteries alone. At 30 days there was an insignificant trend toward decreased thrombogenicity in the FG/FGF-1/heparin treated injured carotids. CONCLUSION: Surface coating with FG/FGF-1/heparin significantly decreases platelet deposition on balloon injured canine carotid arteries after 2 hours of perfusion and may be clinically applicable in endarterectomy and angioplasty procedures. The long-term induction of reendothelialization of arterial surfaces by this technique is under investigation.
Assuntos
Anticoagulantes/farmacologia , Adesivo Tecidual de Fibrina/farmacologia , Fatores de Crescimento de Fibroblastos/fisiologia , Heparina/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Animais , Cateterismo , Cães , Endotélio Vascular , Agregação Plaquetária/fisiologiaRESUMO
PURPOSE: Intimal hyperplasia plagues all types of vascular intervention. Early confluent re-endothelialization may attenuate the smooth muscle cell (SMC) proliferative response. We previously reported that fibroblast growth factor type 1 (FGF-1) and heparin at relative concentrations of 10 ng/ml:250 U/ml delivered in a fibrin glue (FG) suspension can selectively stimulate endothelial cells (EC) and inhibit SMC proliferation in cell culture. This current study evaluates this surface treatment with and without seeded autologous ECs on intimal hyperplasia in a canine carotid artery balloon injury model. METHODS: Twenty-nine adult dogs underwent bilateral balloon injury to a 6 cm segment of their carotid arteries. The injury resulted in a reproducible removal of the intima and 4 to 6 medial lamellae. Nine dogs were used in part I to determine the percent retention of FGF-1 and EC when applied in a FG suspension to the balloon-injured carotid arteries. Part 2 used the remaining 20 dogs to determine the effect of this surface treatment on intimal hyperplasia. In 10 group I dogs, FG (fibrinogen 32.1 mg/ml and thrombin 0.32 U/ml) containing FGF-1 (11 ng/ml) and heparin (250 U/ml) was applied to the luminal surface of one carotid artery, whereas the contralateral carotid artery underwent balloon injury alone. In 10 group II dogs, an identical FG preparation with FGF-1 and heparin was applied to the surface of one carotid artery, whereas the contralateral carotid artery received FG/FGF-1/heparin that also contained autologous ECs (P3; 5 x 10(4) to 10 x 10(4) cells/cm2). Five dogs from both group I and group II were killed at 10 days and the remaining 10 dogs at 30 days. Histologic analysis and computerized morphometric analysis were used to determine intimal and medial thickness and area, percent endothelialization, and medial SMC proliferative rate. RESULTS: There was no measurable neointima in any 10-day dog. There was no difference in neointimal area between the treatments in group I 30-day dogs. There was a significant decrease in maximal neointimal area, intima/media thickness ratio, and intima/media area ratio in group II 30-day dogs that were treated with FG/FGF-1/heparin plus EC. There was an insignificant increase in percent EC coverage and an insignificant decrease in medial SMC proliferative rate in group II 10-day dogs treated with FG/FGF-1/heparin plus EC. CONCLUSIONS: In this canine carotid model, FG with FGF-1 and heparin did not induce significant intimal or medial thickening after 10 or 30 days when compared with vessels that were only balloon-injured. The seeding of autologous ECs within the FG/FGF-1/heparin suspension caused a reduction in neointima formation with no concomitant medial thickening 30 days after injury. The use of FG to locally deliver FGF-1 and ECs may have clinical relevance in the inhibition of intimal hyperplasia.
Assuntos
Artérias Carótidas/efeitos dos fármacos , Lesões das Artérias Carótidas , Cateterismo/efeitos adversos , Endotélio Vascular/citologia , Adesivo Tecidual de Fibrina/uso terapêutico , Fator 1 de Crescimento de Fibroblastos/uso terapêutico , Heparina/uso terapêutico , Túnica Íntima/efeitos dos fármacos , Animais , Artérias Carótidas/patologia , Cateterismo/instrumentação , Células Cultivadas , Modelos Animais de Doenças , Cães , Combinação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Endotélio Vascular/transplante , Hiperplasia/patologia , Hiperplasia/prevenção & controle , Fatores de Tempo , Túnica Íntima/patologiaRESUMO
PURPOSE: The purpose of this study was to report our results in the surgical management of recurrent carotid stenosis (RCS) after carotid endarterectomy (CEA). METHODS: In a 20-year period, we performed 1209 CEAs; 82 operations (6.8%) were for RCS. There were 33 men and 36 women, with an average age of 66.3 years. Nine patients underwent two redo CEAs and two patients underwent three redo CEAs for either bilateral recurrence or a second recurrence on the same side. Overall, 10 patients were identified with a second recurrence. RESULTS: The average time to presentation with RCS was 65 months (range, 3 to 361 months). The majority of patients (66%) were symptomatic, 34% had transient ischemic attacks, 17% had amaurosis fugax, 9% had strokes, and 6% had nonhemispheric symptoms. Before repair, angiograms were obtained. Patch repair was performed in 61 procedures (74%), 41 with vein, 11 with Dacron, and nine with polytetrafluoroethylene. Autogenous or synthetic bypass grafts were used in 20 procedures (24%), vein in eight, Dacron in two, and polytetrafluoroethylene in 10. In one patient, an occluded internal carotid artery was ligated and an endarterectomy of the external carotid artery was performed without a patch. The operative stroke rate was 4.8%. Minor complications included transient or permanent cranial nerve deficits in 7.3% and wound hematomas in 2.4%. CONCLUSION: Although repeat endarterectomy to treat RCS is technically more demanding, it can be performed safely. Long-term follow-up examination shows that a second recurrence may develop, and we recommend serial noninvasive testing.
Assuntos
Estenose das Carótidas/cirurgia , Endarterectomia das Carótidas , Idoso , Prótese Vascular , Artéria Carótida Externa/diagnóstico por imagem , Artéria Carótida Externa/cirurgia , Artéria Carótida Interna/diagnóstico por imagem , Artéria Carótida Interna/cirurgia , Estenose das Carótidas/complicações , Estenose das Carótidas/diagnóstico por imagem , Estenose das Carótidas/mortalidade , Intervalo Livre de Doença , Endarterectomia das Carótidas/efeitos adversos , Feminino , Seguimentos , Humanos , Tábuas de Vida , Masculino , Pessoa de Meia-Idade , Polietilenotereftalatos , Complicações Pós-Operatórias/epidemiologia , Recidiva , Reoperação/efeitos adversos , Fatores de Tempo , Ultrassonografia Doppler em CoresRESUMO
Transinterstitial blood loss at implantation and the degree of graft incorporation and inner capsule thickening was compared in serial explants of albumin-coated Dacron versus blood preclotted Dacron grafts in the canine thoracoabdominal aortic position (8 mm internal diameter x 30 cm length). The coated grafts were Bard DeBakey Vasculour II knitted Dacron prostheses impregnated with carbodiimide-cross-linked human albumin. Control grafts were otherwise identical and preclotted with the recipients' whole blood before heparinization during surgery. Transinterstitial blood loss after establishing flow was measured by weighing sponges wrapped around the grafts. Albumin pretreatment resulted in significantly less median blood loss (5.1 g versus 11 g, P=0.04; Mann-Whitney rank sum test). Grafts were explanted at 1 week, 4 weeks, 10 weeks, and 20 weeks. Patency was 100% in both groups. Graft incorporation at explantation was graded by the surgeon as: 1 = none, 2 = minimal, 3 = moderate, or 4 = extensive. No significant differences were noted at any time period. Inner capsule thickness measurements were made every 2.5 mm along the length of all explants. Grafts explanted at 1 week displayed no inner capsules. By 20 weeks, median inner capsule thickness was significantly less in albumin-coated grafts (190 microm versus 235 microm; P<0.0001). These inner capsules in both groups formed as islands, containing abundant myofibroblasts and collagen, covered by endothelial cells and surrounded by residual fibrin coagula. In conclusion, albumin-coated knitted Dacron grafts displayed less transinterstitial blood loss at implantation, and qualitatively similar incorporation, but significantly thinner inner capsules at 20 weeks.