RESUMO
PURPOSE OF REVIEW: To provide a highlight of the current state of cell therapy for the treatment of critical limb ischemia in patients with diabetes. RECENT FINDINGS: The global incidence of diabetes is constantly growing with consequent challenges for healthcare systems worldwide. In the UK only, NHS costs attributed to diabetic complications, such as peripheral vascular disease, amputation, blindness, renal failure, and stroke, average £10 billion each year, with cost pressure being estimated to get worse. Although giant leaps forward have been registered in the scope of early diagnosis and optimal glycaemic control, an effective treatment for critical limb ischemia is still lacking. The present review aims to provide an update of the ongoing work in the field of regenerative medicine. Recent advancements but also limitations imposed by diabetes on the potential of the approach are addressed. In particular, the review focuses on the perturbation of non-coding RNA networks in progenitor cells and the possibility of using emerging knowledge on molecular mechanisms to design refined protocols for personalized therapy. The field of cell therapy showed rapid progress but has limitations. Significant advances are foreseen in the upcoming years thanks to a better understanding of molecular bottlenecks associated with the metabolic disorders.
Assuntos
Diabetes Mellitus , Angiopatias Diabéticas , Amputação Cirúrgica , Terapia Baseada em Transplante de Células e Tecidos , Angiopatias Diabéticas/terapia , Humanos , Isquemia/terapia , Resultado do TratamentoRESUMO
Cell therapy represents a promising option for revascularization of ischemic tissues. However, injection of dispersed cells is not optimal to ensure precise homing into the recipient's vasculature. Implantation of cell-engineered scaffolds around the occluded artery may obviate these limitations. Here, we employed the synthetic polymer polycaprolactone for fabrication of 3D woodpile- or channel-shaped scaffolds by a computer-assisted writing system (pressure assisted micro-syringe square), followed by deposition of gelatin (GL) nanofibers by electro-spinning. Scaffolds were then cross-linked with natural (genipin, GP) or synthetic (3-glycidyloxy-propyl-trimethoxy-silane, GPTMS) agents to improve mechanical properties and durability in vivo. The composite scaffolds were next fixed by crown inserts in each well of a multi-well plate and seeded with adventitial progenitor cells (APCs, 3 cell lines in duplicate), which were isolated/expanded from human saphenous vein surgical leftovers. Cell density, alignment, proliferation and viability were assessed 1 week later. Data from in vitro assays showed channel-shaped/GPTMS-crosslinked scaffolds confer APCs with best alignment and survival/growth characteristics. Based on these results, channel-shaped/GPTMS-crosslinked scaffolds with or without APCs were implanted around the femoral artery of mice with unilateral limb ischemia. Perivascular implantation of scaffolds accelerated limb blood flow recovery, as assessed by laser Doppler or fluorescent microspheres, and increased arterial collaterals around the femoral artery and in limb muscles compared with non-implanted controls. Blood flow recovery and perivascular arteriogenesis were additionally incremented by APC-engineered scaffolds. In conclusion, perivascular application of human APC-engineered scaffolds may represent a novel option for targeted delivery of therapeutic cells in patients with critical limb ischemia.
Assuntos
Arteriopatias Oclusivas/terapia , Artérias/crescimento & desenvolvimento , Doença Arterial Periférica/patologia , Doença Arterial Periférica/terapia , Transplante de Células-Tronco/instrumentação , Alicerces Teciduais , Túnica Adventícia/citologia , Animais , Arteriopatias Oclusivas/patologia , Artérias/patologia , Células Cultivadas , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Camundongos , Neovascularização Fisiológica/fisiologia , Implantação de Prótese/instrumentação , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos , Resultado do TratamentoRESUMO
Regenerative medicine holds great promise as a way of addressing the limitations of current treatments of ischaemic disease. In preclinical models, transplantation of different types of stem cells or progenitor cells results in improved recovery from ischaemia. Furthermore, experimental studies indicate that cell therapy influences a spectrum of processes, including neovascularization and cardiomyogenesis as well as inflammation, apoptosis and interstitial fibrosis. Thus, distinct strategies might be required for specific regenerative needs. Nonetheless, clinical studies have so far investigated a relatively small number of options, focusing mainly on the use of bone marrow-derived cells. Rapid clinical translation resulted in a number of small clinical trials that do not have sufficient power to address the therapeutic potential of the new approach. Moreover, full exploitation has been hindered so far by the absence of a solid theoretical framework and inadequate development plans. This article reviews the current knowledge on cell therapy and proposes a model theory for interpretation of experimental and clinical outcomes from a pharmacological perspective. Eventually, with an increased association between cell therapy and traditional pharmacotherapy, we will soon need to adopt a unified theory for understanding how the two practices additively interact for a patient's benefit.
Assuntos
Doenças Cardiovasculares/terapia , Medicina Regenerativa/métodos , Transplante de Células-Tronco/métodos , Alquimia , Animais , Transplante de Medula Óssea/métodos , Doenças Cardiovasculares/fisiopatologia , Ensaios Clínicos como Assunto , Humanos , Modelos Teóricos , Preparações Farmacêuticas/administração & dosagem , Farmacologia , Resultado do TratamentoRESUMO
Although definition and mechanistic understanding of pro-angiogenic progenitor cells remains unsatisfactory, general agreement highlights their role in regenerative process following tissue injury and ischemia. Furthermore, stem-cell based therapy represents a hot topic of cardiovascular medicine. Recent studies provide new insights on the signalling pathways that modulate stem/progenitor cell mobilization from bone marrow, homing to ischemic area and participation in vascular remodelling and tissue healing. This review focuses on current knowledge and emerging concepts on stem cell/progenitor cell trafficking in relation to changes in surrounding environment and epigenetic modifications caused by risk factors and comorbidities.
Assuntos
Moléculas de Adesão Celular/fisiologia , Movimento Celular/fisiologia , Citocinas/fisiologia , Neovascularização Fisiológica , Células-Tronco/citologia , Animais , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/terapia , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Quimiocinas/imunologia , Quimiocinas/metabolismo , Quimiocinas/fisiologia , Citocinas/imunologia , Citocinas/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Humanos , Neovascularização Fisiológica/genética , Transdução de Sinais , Transplante de Células-Tronco , Células-Tronco/imunologia , Células-Tronco/metabolismoRESUMO
BACKGROUND AND PURPOSE: Statins, a major component of the prevention of cardiovascular disease, aid progenitor cell functions in vivo and in vitro. Statins bearing a NO-releasing moiety were developed for their enhanced anti-inflammatory/anti-thrombotic properties. Here, we investigated if the NO-donating atorvastatin (NCX 547) improved the functions of circulating angiogenic cells (CACs). EXPERIMENTAL APPROACH: Circulating angiogenic cells (CACs) were prepared from peripheral blood monocytes of healthy volunteers and type-2 diabetic patients and were cultured in low (LG) or high glucose (HG) conditions, in presence of atorvastatin or NCX 547 (both at 0.1 µM) or vehicle. Functional assays (outgrowth, proliferation, viability, senescence and apoptosis) were performed in presence of the endothelial NOS inhibitor L-NIO, the NO scavenger c-PTIO or vehicle. KEY RESULTS: Culturing in HG conditions lowered NO in CACs, inhibited outgrowth, proliferation, viability and migration, and induced cell senescence and apoptosis. NCX 547 fully restored NO levels and functions of HG-cultured CACs, while atorvastatin prevented only apoptosis in CACs. The activity of Akt, a pro-survival kinase, was increased by atorvastatin in LG-cultured but not in HG-cultured CACs, whereas NCX 547 increased Akt activity in both conditions. L-NIO partially blunted and c-PTIO prevented NCX 547-induced improvements in CAC functions. Finally, NCX 547 improved outgrowth and migration of CACs prepared from patients with type 2 diabetes. CONCLUSIONS AND IMPLICATIONS: NCX 547 was more effective than atorvastatin in preserving functions of CACs. This property adds to the spectrum of favourable actions that would make NO-releasing statins more effective agents for treating cardiovascular disease.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Nitratos/farmacologia , Doadores de Óxido Nítrico/farmacologia , Pirróis/farmacologia , Células-Tronco/efeitos dos fármacos , Idoso , Anticolesterolemiantes/farmacologia , Apoptose/efeitos dos fármacos , Benzoatos/farmacologia , Fármacos Cardiovasculares/farmacologia , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Feminino , Humanos , Imidazóis/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Ornitina/análogos & derivados , Ornitina/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
BACKGROUND: Human tissue kallikrein (hK1) generates vasodilator kinins from kininogen and promotes angiogenesis by kinin-dependent and kinin-independent mechanisms. Here, we investigate the expression and functional relevance of hK1 in human gastrointestinal stromal tumour (GIST). METHODS: Vascularisation and hK1 expression of GIST samples were assessed by immunohistochemistry. In two GIST cell lines, hK1 expression was assessed by PCR, and hK1 protein levels and activity were measured by ELISA and an amidolytic assay, respectively. The effect of hK1 silencing, inhibition or overexpression on GIST cell proliferation, migration and paracrine induction of angiogenesis was studied. Finally, local and systemic levels of hK1 were assessed in mice injected with GIST cells. RESULTS: Human tissue kallikrein was detected in 19 out of 22 human GIST samples. Moreover, GIST cells express and secrete active hK1. Titration of hK1 demonstrated its involvement in GIST invasive behaviour, but not proliferation. Furthermore, hK1 released by GIST cells promoted endothelial cell migration and network formation through kinin-dependent mechanisms. Gastrointestinal stromal tumour implantation in nude mice resulted in local and systemic hK1 expression proportional to tumour dimension. CONCLUSIONS: Human tissue kallikrein is produced and released by GIST and participates in tumour invasion. Further studies are needed to validate hK1 as a diagnostic biomarker and therapeutic target in GIST.
Assuntos
Tumores do Estroma Gastrointestinal/metabolismo , Tumores do Estroma Gastrointestinal/patologia , Invasividade Neoplásica , Calicreínas Teciduais/fisiologia , Animais , Linhagem Celular Tumoral , Movimento Celular , Feminino , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Transplante de Neoplasias , Calicreínas Teciduais/sangue , Calicreínas Teciduais/metabolismoRESUMO
Neurotrophins (NTs) control neuron survival and regeneration. Recent research showed that NTs possess cardiovascular actions. In this study, we investigated the hypothesis that the NT nerve growth factor (NGF) prevents cardiomyocyte apoptosis. We demonstrated that cultured rat neonatal cardiomyocytes (RNCMs) produce NGF and express its trkA (tropomyosin-related receptor A (NGF high-affinity receptor)) receptor. RNCMs given a neutralizing antibody for NGF or the trkA inhibitor K252a underwent apoptosis, thus suggesting that NGF is an endogenous prosurvival factor for cardiomyocytes. Adenovirus (Ad)-mediated NGF overexpression protected RNCMs from apoptosis induced by either hypoxia/reoxygenation or angiotensin II (AngII). Similarly, recombinant NGF inhibited AngII-induced apoptosis in isolated rat adult cardiomyocytes. Finally, in a rat model of myocardial infarction, NGF gene transfer promoted cardiomyocyte survival. In RNCMs, recombinant NGF induced trkA phosphorylation, followed by Ser473 phosphorylation and nuclear translocation of phospho-protein kinase B (Akt). In response to Akt activation, Forkhead transcription factors Foxo-3a and Foxo-1 were phosphorylated and excluded from the nucleus. The prosurvival effect of adenoviral vector carrying the human NGF gene was inhibited in vitro by K252a, LY294002 (a pan-phosphatidyl inositol 3-kinase - PI3K - inhibitor), an Akt small interfering RNA, and adenoviruses carrying a dominant negative mutant form of Akt (Ad.DN.Akt) or an Akt-resistant Foxo-3a (Ad.AAA-Foxo-3a). These results newly demonstrate the cardiac prosurvival action of NGF and provide mechanistic information on the signaling pathway, which encompasses trkA, PI3K-Akt, and Foxo.
Assuntos
Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fator de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/metabolismo , Receptor trkA/metabolismo , Angiotensina II/metabolismo , Animais , Apoptose/efeitos dos fármacos , Carbazóis/farmacologia , Sobrevivência Celular , Células Cultivadas , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Humanos , Alcaloides Indólicos/farmacologia , Morfolinas/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Fator de Crescimento Neural/genética , Proteínas do Tecido Nervoso/metabolismo , Células PC12 , Fosforilação , Ratos , Transdução de Sinais , TransfecçãoRESUMO
BACKGROUND AND PURPOSE: Mature endothelial cells and their progenitors are dysfunctional in diabetes, resulting in deficient neovascularisation following arterial occlusion. This study aimed to evaluate the therapeutic activity of a nitric oxide (NO) releasing statin in the setting of experimental diabetes and peripheral ischaemia. EXPERIMENTAL APPROACH: The effects of NCX 6550, an NO-releasing pravastatin derivative, on angiogenesis in ischaemic limbs was studied in normoglycaemic mice or mice made diabetic by treatment with streptozotocin (STZ). Control mice received an equimolar dosage of the parent statin compound, pravastatin. The therapeutic action of NCX 6550 was also tested in mice lacking the gene for endothelial nitric oxide synthase (eNOS). KEY RESULTS: In normoglycaemic or STZ-diabetic CD1 mice, only NCX 6550 stimulated skeletal muscle revascularisation. In addition, NCX 6550 induced greater improvement in limb reperfusion and salvage, than pravastatin. The number of circulating endothelial progenitor cells was decreased in STZ-diabetic mice, this defect being prevented by NCX 6550 and, to a lesser extent by pravastatin. In vitro, high glucose concentrations reduced the migratory capacity of endothelial progenitor EPCs, which was partly reversed by preincubation with pravastatin and completely reversed by NCX 6550. The postischaemic recovery of eNOS knockout mice was severely impaired as a consequence of depressed angiogenesis and this recovery was improved by treatment with NCX 6550, but not with pravastatin. CONCLUSIONS AND IMPLICATIONS: These findings indicate that incorporation of a bioactive NO moiety improves the therapeutic profile of statins for the treatment of peripheral vascular disease.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Isquemia/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Nitratos/uso terapêutico , Pravastatina/análogos & derivados , Pravastatina/uso terapêutico , Animais , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Membro Posterior/irrigação sanguínea , Hipertensão/tratamento farmacológico , Hipertensão/metabolismo , Hipertensão/patologia , Isquemia/metabolismo , Isquemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/deficiência , Óxido Nítrico Sintase Tipo III/genéticaRESUMO
Microfabrication techniques were combined with fractal algorithms to realize polymeric scaffolds resembling capillary networks. The scaffolds were seeded with human endothelial cells in monoculture as well as in coculture with human fibroblasts. To enhance the process of angiogenesis, endothelial cells were transfected with an adenoviral vector carrying the gene for human tissue kallikrien. The results demonstrate that both the presence of a structured scaffold as well as fibroblasts in coculture contribute synergically to the promotion of a metabolically active network. The fractal scaffolds have several possible applications for example in vascularized tissue engineering and therapeutic angiogenesis. A broader implication of these results is that cell-extra cellular matrix and cell-cell interactions cooperate dynamically both at a biochemical as well as microstructural level.
Assuntos
Materiais Biocompatíveis/química , Capilares/crescimento & desenvolvimento , Neovascularização Fisiológica , Polímeros/química , Engenharia Tecidual/métodos , Algoritmos , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Fractais , Vetores Genéticos , Humanos , Teste de Materiais , Poliésteres/química , Sais de Tetrazólio , Tiazóis , Calicreínas Teciduais/genética , TransfecçãoRESUMO
Ischemic disease represents the new epidemic worldwide. Animal models of ischemic disease are useful because they can help us to understand the underlying pathogenetic mechanisms and develop new therapies. The present review article summarizes the results of a consensus conference on the status and future development of experimentation in the field of cardiovascular medicine using murine models of peripheral and myocardial ischemia. The starting point was to recognize the limits of the approach, which mainly derive from species- and disease-related differences in cardiovascular physiology. For instance, the mouse heart beats at a rate 10 times faster than the human heart. Furthermore, healing processes are more rapid in animals, as they rely on mechanisms that may have lost relevance in man. The main objective of the authors was to propose general guidelines, diagnostic end points and relevance to clinical problems.
Assuntos
Experimentação Animal , Modelos Animais de Doenças , Extremidades/irrigação sanguínea , Oclusão de Enxerto Vascular/fisiopatologia , Isquemia/fisiopatologia , Isquemia Miocárdica/fisiopatologia , Experimentação Animal/ética , Experimentação Animal/legislação & jurisprudência , Animais , Aterosclerose/cirurgia , Comorbidade , Consenso , Diabetes Mellitus Tipo 1/fisiopatologia , Determinação de Ponto Final , Oclusão de Enxerto Vascular/terapia , Guias como Assunto , Humanos , Isquemia/terapia , Camundongos , Isquemia Miocárdica/terapia , Medicina Regenerativa , Reprodutibilidade dos Testes , Índice de Gravidade de Doença , Especificidade da Espécie , Veias/transplante , CicatrizaçãoRESUMO
AIMS/HYPOTHESIS: Benfotiamine, a vitamin B1 analogue, reportedly prevents diabetic microangiopathy. The aim of this study was to evaluate whether benfotiamine is of benefit in reparative neovascularisation using a type I diabetes model of hindlimb ischaemia. We also investigated the involvement of protein kinase B (PKB)/Akt in the therapeutic effects of benfotiamine. METHODS: Streptozotocin-induced diabetic mice, given oral benfotiamine or vehicle, were subjected to unilateral limb ischaemia. Reparative neovascularisation was analysed by histology. The expression of Nos3 and Casp3 was evaluated by real-time PCR, and the activation state of PKB/Akt was assessed by western blot analysis and immunohistochemistry. The functional importance of PKB/Akt in benfotiamine-induced effects was investigated using a dominant-negative construct. RESULTS: Diabetic muscles showed reduced transketolase activity, which was corrected by benfotiamine. Importantly, benfotiamine prevented ischaemia-induced toe necrosis, improved hindlimb perfusion and oxygenation, and restored endothelium-dependent vasodilation. Histological studies revealed the improvement of reparative neovascularisation and the inhibition of endothelial and skeletal muscle cell apoptosis. In addition, benfotiamine prevented the vascular accumulation of advanced glycation end products and the induction of pro-apoptotic caspase-3, while restoring proper expression of Nos3 and Akt in ischaemic muscles. The benefits of benfotiamine were nullified by dominant-negative PKB/Akt. In vitro, benfotiamine stimulated the proliferation of human EPCs, while inhibiting apoptosis induced by high glucose. In diabetic mice, the number of circulating EPCs was reduced, with the deficit being corrected by benfotiamine. CONCLUSIONS/INTERPRETATION: We have demonstrated, for the first time, that benfotiamine aids the post-ischaemic healing of diabetic animals via PKB/Akt-mediated potentiation of angiogenesis and inhibition of apoptosis. In addition, benfotiamine combats the diabetes-induced deficit in endothelial progenitor cells.
Assuntos
Apoptose/efeitos dos fármacos , Angiopatias Diabéticas/tratamento farmacológico , Isquemia/tratamento farmacológico , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica/efeitos dos fármacos , Tiamina/análogos & derivados , Animais , Western Blotting , Peso Corporal , Caspase 3 , Inibidores de Caspase , Caspases/metabolismo , Proliferação de Células/efeitos dos fármacos , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/fisiopatologia , Angiopatias Diabéticas/fisiopatologia , Suplementos Nutricionais , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Células Endoteliais/fisiologia , Ativação Enzimática/efeitos dos fármacos , Hemodinâmica/efeitos dos fármacos , Imuno-Histoquímica , Isquemia/fisiopatologia , Isquemia/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Distribuição Aleatória , Células-Tronco/efeitos dos fármacos , Células-Tronco/patologia , Células-Tronco/fisiologia , Tiamina/farmacologia , Tiamina/uso terapêuticoRESUMO
Human pancreatic islet transplantation has recently been shown to be successful in replacing pancreatic endocrine function into type 1 diabetic recipients. A major drawback, however, is the high amount of pancreatic ss cells required to render one single patient insulin-independent. Given the shortage of human beta cell donors, the majority of type 1 diabetic patients remain excluded from this therapeutic option. High number of islets are requested since substantial islet cell death and dysfunction occur within the first few hours and days after islet transplantation. Impaired vascularization of the engraft, the non-specific inflammatory reaction at the site of transplantation, together with the presence of active or memory autoimmune responses to islet autoantigens and allogeneic recognition contribute to apoptosis of ss cells and subsequent early graft function loss. This review will focus on ex vivo engineering of the islet graft by gene transfer to improve islet engraftment. An overview of currently used gene transfer techniques will be given and their potential will be discussed.
Assuntos
Terapia Genética , Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/fisiologia , Animais , Diabetes Mellitus Tipo 1/terapia , Vetores Genéticos , Sobrevivência de Enxerto , HumanosRESUMO
BACKGROUND: In systemic sclerosis (SSc) the lack of an angiogenic response to hypoxia may be due to inappropriate synthesis of angiogenic and angiostatic factors. Tissue kallikrein (t-kallikrein), regulating the kallikrein-kinin system and acting on the microcirculation, is a potent angiogenic agent, and kallistatin is its natural inhibitor. OBJECTIVE: To evaluate, in patients with SSc, t-kallikrein and kallistatin levels and their correlation with clinical features and measures of microvascular involvement. PATIENTS AND METHODS: Serum levels of t-kallikrein and kallistatin (ELISA) and t-kallikrein skin expression (immunohistochemistry) were studied in patients with SSc, and evaluated for subset (dSSc or lSSc), clinical and immunological features, and microvascular involvement (ulcers, telangiectasias, nailfold videocapillaroscopy). RESULTS: Circulating levels of t-kallikrein were higher in SSc than in controls (p<0.001). T-kallikrein did not differ between lSSc and dSSc, although it was higher in lSSc than in controls (p<0.001).T-kallikrein levels were higher in patients with early and active capillaroscopic pattern than in those with late pattern (p = 0.019 and 0.023). Patients with giant capillaries and capillary microhaemorrhages had higher t-kallikrein concentrations than patients with architectural derangement (p = 0.04). No differences in kallistatin levels were detected between patients with SSc and controls, or between lSSc and dSSc. In early SSc skin, the presence of t-kallikrein was found in endothelial and in perivascular inflammatory cells, while no staining in skin of advanced SSc was detected. CONCLUSION: T-kallikrein levels are increased in patients with SSc, particularly in lSSc, and are associated with early and active capillaroscopic patterns. T-kallikrein may play a part in SSc microvascular changes.
Assuntos
Escleroderma Sistêmico/sangue , Calicreínas Teciduais/sangue , Adulto , Idoso , Autoanticorpos/sangue , Capilares/patologia , Proteínas de Transporte/sangue , Endotélio Vascular/metabolismo , Feminino , Humanos , Técnicas Imunoenzimáticas , Masculino , Microcirculação , Angioscopia Microscópica/métodos , Pessoa de Meia-Idade , Escleroderma Sistêmico/patologia , Serpinas/sangue , Pele/metabolismo , Calicreínas Teciduais/antagonistas & inibidoresRESUMO
Angiogenesis is essential for the repair of wounds and tissues damaged by ischemia. The regenerative process is tightly regulated by master angiogenic factors, cytokines and the downstream mediator NO. In addition, modulators of vascular growth, such as COX-2-generated prostanoids, contribute to the process by stabilizing the hypoxia-inducible factor and stimulating the expression of VEGF. Recently, we discovered that human tissue kallikrein, a member of the serine proteinase superfamily, possesses potent angiogenic effects. It has been categorized as a pleiotropic angiogenic agent acting via enzymatic cleavage of kininogen and subsequent release of kinin peptides. Kinins bind G-protein coupled receptors, subtype B1 and B2, and exert proliferative effects on endothelial cells via an IP3K-Akt-NO mediated mechanism independent of VEGF. In addition, kinins stimulate the release of angiogenic prostacyclin. Gene transfer of human tissue kallikrein rescues ischemic tissues in otherwise normal mice, as well as in hypertensive or diabetic animals. In addition, prophylactic gene delivery of tissue kallikrein to diabetic skeletal muscles prevents the development of microangiopathy and stimulates collateralization, thus protecting from the consequences of supervening arterial occlusion.
Assuntos
Coagulantes/farmacologia , Coagulantes/uso terapêutico , Técnicas de Transferência de Genes , Calicreínas/farmacologia , Calicreínas/uso terapêutico , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Complicações do Diabetes , Modelos Animais de Doenças , Humanos , Hipertensão/complicações , Calicreínas/genética , Camundongos , Isquemia Miocárdica/terapia , Isquemia Miocárdica/veterinária , Acidente Vascular Cerebral/terapia , Fator A de Crescimento do Endotélio VascularRESUMO
AIMS/HYPOTHESIS: Type 1 diabetes increases the risk of peripheral ischaemia and impairs recovery once ischaemia occurs, probably because the healing process is hampered by diabetes-induced endothelial dysfunction. In normoglycaemic mice subjected to limb ischaemia, blockade of nerve growth factor (NGF) compromises reparative angiogenesis. In the present study, we evaluated if expressional alterations of endogenous NGF system components are associated with diabetes-related impairment in neovascularisation. In addition, we tested whether the correction of NGF liabilities benefits post-ischaemic healing of Type 1 diabetic animals. METHODS: Unilateral hindlimb ischaemia was produced in streptozotocin-induced Type 1 diabetic mice. Purified murine NGF (20 microg daily for 14 days) or PBS were injected into ischaemic adductors. Non-diabetic mice given PBS served as controls. Hindlimb blood flow was analysed sequentially for up to 14 days. At necroscopy, adductors were removed for quantification of microvessel density, endothelial cell apoptosis and NGF receptor expression. NGF content was determined by ELISA three days after ischaemia. In vitro, we tested whether NGF protects endothelial cells from apoptosis induced by high glucose and whether vascular endothelial growth factor-A (VEGF-A) is involved in this beneficial effect. RESULTS: Muscles removed from Type 1 diabetic mice showed reduced NGF content and up-regulation of the NGF p75 receptor. NGF supplementation promoted capillarisation and arteriogenesis, reduced apoptosis, and accelerated blood flow recovery. NGF stimulated VEGF-A production by human endothelial cells incubated in high-glucose medium and conferred resistance against high-glucose-induced apoptosis via a VEGF-A-mediated mechanism. CONCLUSIONS/INTERPRETATION: NGF protects endothelial cells from apoptosis induced by Type 1 diabetes and facilitates reparative neovascularisation. The findings may open up new therapeutic options for the treatment of diabetic complications.
Assuntos
Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/tratamento farmacológico , Membro Posterior/efeitos dos fármacos , Membro Posterior/lesões , Isquemia/complicações , Isquemia/tratamento farmacológico , Fator de Crescimento Neural/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Capilares/efeitos dos fármacos , Capilares/patologia , Capilares/fisiopatologia , Sobrevivência Celular , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 1/fisiopatologia , Esquema de Medicação , Avaliação Pré-Clínica de Medicamentos/métodos , Endotélio Vascular/lesões , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Expressão Gênica , Membro Posterior/irrigação sanguínea , Isquemia/fisiopatologia , Masculino , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , Músculo Esquelético/ultraestrutura , Neovascularização Patológica/genética , Neovascularização Patológica/fisiopatologia , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/fisiologia , Fator de Crescimento Neural/antagonistas & inibidores , Fator de Crescimento Neural/genética , Receptor de Fator de Crescimento Neural , Receptores de Fator de Crescimento Neural/antagonistas & inibidores , Receptores de Fator de Crescimento Neural/efeitos dos fármacos , Receptores de Fator de Crescimento Neural/genética , Reperfusão , Vasos Retinianos/fisiologia , Vasos Retinianos/ultraestrutura , Veias Umbilicais/citologia , Veias Umbilicais/efeitos dos fármacos , Veias Umbilicais/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
AIMS/HYPOTHESIS: The neurotrophin nerve growth factor (NGF) is pro-angiogenic and facilitates wound repair. The present study was conducted to (i) assess the statement of NGF system components in diabetic wounds and (ii) evaluate whether NGF supplementation could prevent impairment of wound neoangiogenesis by diabetes. METHODS: Skin wounds were produced in the interscapular region of streptozotocin-induced diabetic mice. NGF (1 microg per day in PBS) or vehicle was applied onto the ulcers for 3 days after punching. Non-diabetic mice were used as controls. RESULTS: In wounds of untreated diabetic mice, endogenous levels of immunoreactive NGF were lower than those in wounds of non-diabetic mice ( p<0.01). Immunohistochemical analysis showed down-regulation of tyrosine kinase receptor-A (TrkA) and up-regulation of p75 receptor in granulation tissue microvasculature. Local NFG administration prevented diabetes-induced expressional alterations, enhanced reparative capillarisation ( p<0.01), and accelerated wound closure ( p<0.01). This was associated with a three-fold increase in endothelial cell proliferation ( p<0.01), while apoptosis was reduced by 50% ( p<0.05). Quantitative RT-PCR documented a 5.5-fold increase in the expression of vascular endothelial growth factor-A (VEGF-A) by exogenous NGF in diabetic tissues ( p<0.01). In in vitro preparations of human endothelial cells from derma, NGF increased the release of immunoreactive VEGF-A, and reduced high-glucose-induced apoptosis ( p<0.05), the latter effect being inhibited by a VEGF-A receptor-2 antagonist. CONCLUSIONS/INTERPRETATION: Diabetic ulcers display distinct alterations in reparative angiogenesis and in the expression of NGF and its receptors. NGF supplementation corrects endogenous liabilities, facilitates vascular regeneration, and suppresses endothelial apoptosis seemingly via VEGF-A. Our findings unravel new mechanisms responsible for NGF reparative action.
Assuntos
Apoptose/efeitos dos fármacos , Diabetes Mellitus Tipo 1/complicações , Células Endoteliais/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Fator de Crescimento Neural/uso terapêutico , Úlcera Cutânea/complicações , Úlcera Cutânea/tratamento farmacológico , Administração Cutânea , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus Experimental/complicações , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Expressão Gênica/efeitos dos fármacos , Glucose/efeitos adversos , Glucose/antagonistas & inibidores , Glucose/metabolismo , Humanos , Itália , Masculino , Camundongos , Neovascularização Fisiológica/fisiologia , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Receptores de Fator de Crescimento Neural/efeitos dos fármacos , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Úlcera Cutânea/patologia , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologiaRESUMO
Kinins, the biological end-products of the kallikrein-kininogen system, influence many aspects of the cellular function. Interest in this peptidergic system has been renewed recently by the discovery that kinins exert cardiovascular protective effects and promote post-ischaemic recovery by stimulating vascular growth. Pharmacological and genetic studies indicate that induction of kallikrein and kinin receptors by ischaemia is functionally relevant in the natural host response that permits perfusion recovery and tissue healing. Furthermore, potentiation of the generation of kinins by continuous supply of tissue kallikrein promotes reparative angiogenesis through stimulation of the release of nitric oxide and prostaglandins. Strategies that activate kinin receptors might be applicable to the treatment of occlusive vascular disease, whereas kinin receptor antagonists could represent therapeutic reagents against pathological angiogenesis in cancer and chronic inflammatory conditions.
Assuntos
Isquemia/terapia , Sistema Calicreína-Cinina , Neovascularização Fisiológica/fisiologia , Receptores de Superfície Celular/fisiologia , Animais , Receptor de Asialoglicoproteína , Humanos , Sistema Calicreína-Cinina/genética , Sistema Calicreína-Cinina/fisiologiaRESUMO
Ischaemic diseases are characterized by an impaired supply of blood resulting from narrowed or blocked arteries that starve tissues of needed nutrients and oxygen. Coronary-atherosclerosis induced myocardial infarction is one of the leading causes of mortality in developed countries. Ischaemic disease also affects the lower extremities. Considerable advances in both surgical bypassing and percutaneous revascularization techniques have been reached. However, many patients cannot benefit from these therapies because of the extension of arterial occlusion and/or microcirculation impairment. Consequently, the need for alternative therapeutic strategies is compelling. An innovative approach consists of stimulating collateral vessel growth, a natural host defence response that intervenes upon occurrence of critical reduction in tissue perfusion (Isner & Asahara, 1999). This review will debate the relevance of therapeutic angiogenesis for promotion of tissue repair. The following issues will receive attention: (a) vascular growth patterns, (b) delivery systems for angiogenesis gene transfer, (c) achievements of therapeutic angiogenesis in myocardial and peripheral ischaemia, and (d) future directions to improve effectiveness and safety of vascular gene therapy.
Assuntos
Terapia Genética/métodos , Isquemia/terapia , Neovascularização Fisiológica , Animais , Previsões , Terapia Genética/tendências , Humanos , Isquemia/genética , Modelos BiológicosRESUMO
Angiogenesis represents a compensatory response targeted to preserve the integrity of tissues subjected to ischemia. The aim of the present study was to examine whether reparative angiogenesis is impaired in spontaneously hypertensive rats (SHR), as a function of progression of hypertension. In addition, the potential of gene therapy with human tissue kallikrein (HK) in revascularization was challenged in SHR and normotensive Wistar-Kyoto rats (WKY) that underwent excision of the left femoral artery. Expression of vascular endothelial growth factor and HK was upregulated in ischemic hindlimb of WKY but not of SHR. Capillary density was increased in ischemic adductor muscle of WKY (from 266+/-20 to 633+/-73 capillaries/mm(2) at 28 days, P<0.001), whereas it remained unchanged in SHR (from 276+/-20 to 354+/-48 capillaries/mm(2), P=NS), thus compromising perfusion recovery as indicated by reduced plantar blood flow ratio (0.61+/-0.08 versus 0.92+/-0.07 in WKY at 28 days, P<0.05). In separate experiments, saline or 5x10(9) pfu adenovirus containing the HK gene (Ad.CMV-cHK) or the beta-galactosidase gene (Ad.CMV-LacZ) was injected intramuscularly at 7 days after the induction of ischemia. Ad.CMV-cHK augmented capillary density and accelerated hemodynamic recovery in both strains, but these effects were more pronounced in SHR (P<0.01). Our results indicate that native angiogenic response to ischemia is impaired in SHR, possibly as a result of defective modulation of endothelial cell mitogens. Supplementation with kallikrein, one of the growth factors found to be deficient in SHR, restores physiological angiogenic response utilitarian for tissue healing. Our discoveries may have important implications in vascular medicine for therapeutic benefit.
Assuntos
Calicreínas/fisiologia , Músculos/fisiologia , Neovascularização Fisiológica/fisiologia , Animais , Técnicas de Transferência de Genes , Membro Posterior/irrigação sanguínea , Humanos , Injeções Intramusculares , Isquemia/genética , Isquemia/fisiopatologia , Calicreínas/genética , Calicreínas/farmacologia , Masculino , Perfusão , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Fluxo Sanguíneo RegionalRESUMO
The involvement of kinins, calcitonin gene-related peptide (CGRP), and tachykinins during mesenteric post-ischemic reperfusion was studied in anesthetized rats by using antagonists for bradykinin (BK) B1, BK B2, CGRP1, or tachykinin NK1 receptor, or by capsaicin-induced desensitization. B1, B2, or CGRP1 receptor antagonists or desensitization attenuated the transient hypotension and plasma protein and leukocyte infiltration of intestinal wall observed during post-ischemic reperfusion. These effects were abolished by the combination of B2 and CGRP1 blockade as well as by B2 antagonism in capsaicinized rats, while NK1 blockade was ineffective. Our results suggest that kinins and CGRP contribute to systemic vasodilatation and microvascular leakage during mesenteric reperfusion. Pharmacological blockade of these systems could help preventing hypotension and intestinal injury consequent to reperfusion.