Methylglyoxal (MG), a highly reactive dicarbonyl accumulates after myocardial infarction (MI), causing adverse remodelling and cardiac dysfunction. We hypothesized that therapy using bone marrow cells (BMCs) overexpressing glyoxalase1 (Glo1), the main enzyme that metabolizes MG, injected into mouse MI model would translate into better survival of transplanted cells and improve their therapeutic effect. We found that Glo1 expression is significantly reduced at 7 days post-MI. Glo1 BMCs exposed to MG in vitro displayed greater angiogenic potential and reduced reactive oxygen species production compared to wild type (WT) BMCs. However, in the mouse MI model, Glo1 BMCs did not improve cardiac function or vascularity or reduce scar formation compared to WT BMCs and saline treatments. In conclusion, Glo1 overexpression in BMCs does not confer superior therapeutic efficacy for treating MI under the conditions tested.

Orientador: Cristina Pontes Vicente / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T21:32:05Z (GMT). No. of bitstreams: 1 Godoy_JulianaAparecidaPretode_M.pdf: 1738296 bytes, checksum: 42d22e3271b69962a476983e12e359ed (MD5) Previous issue date: 2010 / Resumo: O dermatan sulfato (DS) é um glicosaminoglicano que pode atuar como um agente antitrombótico, anticoagulante e anti-inflamatório. A aterosclerose é uma doença que acomete os vasos sanguíneos através da formação exacerbada de placas de gordura, interrompendo o fluxo de sangue. A intervenção cirúrgica mais utilizada nesses casos é a angioplastia, entretanto, esse procedimento pode ocasionar uma lesão ao endotélio, onde células e proteínas inflamatórias são recrutadas ao local da lesão, promovendo a migração e proliferação das células musculares lisas ocasionando a reestenose (neointima) do vaso acometido. A recuperação do endotélio, momentos após a lesão, seria uma alternativa terapêutica com o objetivo de se evitar a formação de neointima. Essa recuperação poderia ser feita pelas células progenitoras endoteliais (EPC), existentes na população de células mononucleares (MNC), da medula óssea. Neste estudo, testamos o efeito do DS na inflamação, trombose, formação de neointima e migração de MNC em camundongos selvagens (C57BL06), testamos também o efeito do DS em conjunto com a administração de MNC na formação de neointima em camundongos selvagens, deficientes da proteína cofator II da heparina (HCII-/-) e deficientes em apolipoproteína E (ApoE-/-). Todos os animais analisados passaram por um procedimento cirúrgico na artéria carótida comum esquerda, mimetizando a lesão causada por angioplastia em humanos. Analisamos a formação de trombo, a presença de células inflamatórias e de P-selectina nos animais 1 ou 3 dias após a lesão arterial; a formação de neointima foi analisada 21 após a injúria arterial. Observamos uma inibição da trombose, diminuição de células CD45+ e da expressão de P-selectina no local da lesão nos animais selvagens tratados com DS. Foi observado, também, que nos animais selvagens que receberam MNCs ou MNCs + DS, a formação de neointima foi inibida. Nos animais selvagens tratados com MNC + DS, houve uma maior migração de MNC para o local da lesão. Nos animais HCII-/-, não houve inibição da formação da neointima em nenhum dos dois grupos (tratados com MNC e tratados com MNC + DS). Nos camundongos ApoE-/- a injeção de MNC mesmo em conjunto com o tratamento com DS não foi capaz de inibir a proliferação de neointima; houve melhora apenas quando se administrou o DS isoladamente. Com isso, concluímos que o DS participa da inibição dos processos trombótico e inflamatório, na sua fase inicial, após injúria arterial e promove também a migração de um número maior de MNCs para o local da lesão nos animais selvagens; nos animais ApoE-/-, o DS inibiu a resposta inflamatória inicial e a formação de neointima apenas quando administrado isoladamente, tendo seu efeito anulado quando este foi injetado conjuntamente com as MNC. Este dado sugere que o processo inflamatório local, o estágio de formação das placas de ateroma, o elevado índice de colesterol e os triglicerídeos circulantes podem influenciar no efeito do DS e na capacidade de recuperação do endotélio mediada pela MNCs injetadas nos camundongos ApoE-/-. / Abstract: Dermatan sulfate (DS) is a glycosaminoglycan that can act as an antithrombotic, anticoagulant and anti-inflammatory agent. Atherosclerosis is a disease that affects the blood vessels by an exacerbated fat plaques formation, blocking the blood flow. The most used surgical operation, in these cases, is the angioplasty; however, this procedure can cause a lesion to the endothelium, where inflammatory cells and proteins are recruited to the lesion site, promoting smooth muscle cells migration and proliferation, provoking restenosis (neointima) of the attempted vessel. The endothelium recovery, some time after lesion, would be a therapeutic strategy by preventing the neointima formation. This recovery could be done by the endothelial progenitor cells (EPC), present in mononuclear cells (MNC) population, from bone marrow. In this study, we devise the DS effect on inflammation, thrombosis, neointima formation and MNC migration in wild-type mice (C57BL06); we also devise DS effect together with MNC administration on neointima formation in wild-type mice, heparin cofactor II deficient mice (HCII-/-) and apolipoprotein E deficient mice (ApoE-/-). All analyzed animals suffered surgical operation in left common carotid artery, that mimics the lesion caused by angioplasty in humans. We analyzed thrombus formation, inflammatory cells presence and P-selectin in animals 1 and 3 days after lesion; neointima formation was analyzed 21 days after arterial injury. We observed a thrombus inhibition, decreased CD45+ cells and P-selectin expression at the lesion site in wild-type animals treated with DS. It was also observed in wild-type animals that received MNC or MNC + DS, the neointima formation was inhibited. In wild-type animals treated with MNC + DS, there were a higher MNC migration to the lesion site. In HCII-/- animals, there was not an inhibition in neointima formation in any of the two groups (treated with MNC or treated with MNC + DS). In ApoE-/- mice, the MNC injection even in DS presence, was not able to inhibit neointima proliferation; there was an improvement only when DS was administered alone. It follows that DS participate on thrombotic and inflammatory process inhibition, in the initial stages, after arterial injury and it also promote a higher MNC migration to the lesion site in wild-type animals; in ApoE-/- animals, DS inhibited the initial inflammatory response and neointima formation only when administered alone; its effect was null when it was injected together with MNC. This date suggest that local inflammatory process, atheroma plaque state, elevated cholesterol and triglyceride rates can influence on DS effect and on the capacity of endothelium recovery mediated by injected MNC in ApoE-/- mice. / Mestrado / Histologia / Mestre em Biologia Celular e Estrutural

The contribution of the bone marrow microenvironment in myelodysplastic syndrome is controversial. We therefore analyzed the functional properties of primary mesenchymal stromal cells from patients with myelodysplastic syndrome in the presence or absence of lenalidomide. Compared to healthy controls, clonality and growth were reduced across all disease stages. Furthermore, differentiation defects and particular expression of adhesion and cell surface molecules (e.g. CD166, CD29, CD146) were detected. Interestingly, the levels of stromal derived factor 1-alpha in patients’ cells culture supernatants were almost 2-fold lower (P<0.01) than those in controls and this was paralleled by a reduced induction of migration of CD34+ hematopoietic cells. Co-cultures of mesenchymal stromal cells from patients with CD34+ cells from healthy donors resulted in reduced numbers of cobblestone area-forming cells and fewer colony-forming units. Exposure of stromal cells from patients and controls to lenalidomide led to a further reduction of stromal derived factor 1-alpha secretion and cobblestone area formation, respectively. Moreover, lenalidomide pretreatment of mesenchymal stromal cells from patients with low but not high-risk myelodysplastic syndrome was able to rescue impaired erythroid and myeloid colony formation of early hematopoietic progenitors. In conclusion, our analyses support the notion that the stromal microenvironment is involved in the pathophysiology of myelodysplastic syndrome thus representing a potential target for therapeutic interventions.

Hematopoietic stem cells (HSCs) reside in the bone marrow (BM) and generate blood cells for the entire lifespan of an animal. HSCs are mostly quiescent, but can self-renew and generate all lineages of the hematopoietic system. Their clinical significance lies in their potential to engraft after transplantation and reconstitute the blood and immune system in patients with hematological malignancies, immune deficiencies or hemoglobin abnormalities. Despite significant progress in our understanding of mechanisms involved in self-renewal, differentiation and quiescence, a coherent picture of how these mechanisms act in concert to regulate steady-state function and homeostatic responses of HSCs has not emerged yet. Importantly, reliable renewal or even maintenance of HSCs in vitro remains challenging. The identification of dozens of cytokines and of more than 200 genes affecting HSC function in knockout studies, as well as multiple publications on genome-wide expression and epigenetic signatures, still leaves significant gaps in our understanding. From a clinical-translational perspective, it is essential to bridge these gaps in our knowledge to devise strategies to maintain HSCs in vitro. This would have enormous implications for the current practice of allogeneic and autologous bone marrow transplantation, as well as gene therapy and genome editing targeting HSCs. Our lab has previously shown that culture in the presence of reduced calcium concentrations allowed striking maintenance of HSC function over at least two weeks. Furthermore, calcium controlled expression of the master hematopoietic tumor suppressor, TET2, while TET2 expression affected the response of HSCs to extracellular calcium. Despite this progress, quantitative expansion of functional HSCs was not achieved through low-calcium culture, suggesting other barriers to self-renewal exist in vitro. The goal of this thesis is to gain a deeper understanding in the barriers to self-renewal of HSCs, both in vitro and in vivo. During fetal life, HSC develop in the fetal liver (FL), where they expand, and home to the BM around birth. As FL HSCs exhibit more self-renewal than adult HSCs, we examined the response of these cells to calcium and to deletion of Tet2 in hopes of identifying barriers to self-renewal in the adult. Surprisingly, we observed that FL HSCs have very distinct calcium physiology compared to adult HSCs and could not be maintained in vitro in any calcium concentration. Only in the presence of low-calcium and after deletion of Tet2 could maintenance of functional FL HSCs be achieved in vitro. This is in sharp contrast to adult HSCs, which were maintained in low-calcium conditions, and in which deletion of Tet2 attenuated maintenance in these conditions. These data indicate more profound differences in the biology of fetal versus adult HSCs than previously appreciated, and suggest that recapitulating the extensive renewal capacity of FL HSCs in adult HSCs may not possible with identical culture conditions. Further studies into mechanisms involved in HSC maintenance in low-calcium conditions revealed that these conditions attenuated the propensity of HSCs to differentiate into megakaryocytes (Mk), hyperploid cells that generate platelets essential to hemostasis. Whereas most hematopoietic lineages arise through successive, increasingly lineage-committed progenitors, Mks can derive rapidly and directly from HSCs. Direct megakaryopoiesis from HSCs occurs in particular in response to inflammatory stimuli, such as interferon signaling. We therefore tested the hypothesis that direct Mk specification is a barrier to HSC self-renewal that is alleviated at least in part by culture in low-calcium conditions. Interferon signaling has been reported to induce direct megakaryopoiesis and also rapidly recruits HSCs into cell cycle. HSCs are also known to be susceptible to replication stress and ensuing DNA damage. We therefore examined the connection between DNA damage responses (DDR) and direct megakaryopoiesis. We discovered that interferon signaling induced DNA damage through replication stress in vivo, whereas irradiation rapidly induced Mk commitment in HSCs. These findings established a connection between a DDR and direct megakaryopoiesis. Furthermore, quiescent HSCs are subject to a physiological DDR caused by hypertranscription, while in vitro culture induced replication stress. Inflicting additional DNA damage in HSCs in vitro or in vivo rapidly induced expression of Mk markers. Even in the absence of additional DNA damage, pharmacological blockade of the G2 phase of the cell cycle induced MK differentiation and hyperploidy in HSCs, but apoptosis in progenitors. Part of the underlying mechanisms are post-transcriptional. Increased protein expression of the Mk lineage transcription factor GATA1 was induced by both DNA damage and G2 arrest, and preceded upregulation of Gata1 mRNA and other Mk genes. Expression of GATA1 protein is at least in part mediated by the integrated stress response (ISR), which modulates translation. Together these findings show that direct megakaryopoiesis from HSCs can be stimulated by DNA damage-induced G2 arrest and is at least partially post-transcriptionally regulated. As our findings suggested that direct megakaryopoiesis, among others induced by a DDR, limits HSC maintenance, we initiated studies to identify the mechanism underlying the DDR in cycling HSCs. We discovered that cycling HSCs are particularly prone to misincorporation of uracil into DNA in vivo and in vitro. Supplementation with thymidine in vitro decreased uracil incorporation, attenuated the DDR, and strikingly increased the maintenance of multipotential HSCs in vitro. Thymidine supplementation also lowered expression of CD41, a marker of Mk-committed HSCs. These data establish a profound role of a uracil-induced DDR in HSCs and indicate that direct commitment to the Mk lineage is inversely correlated with functional HSC maintenance. The DDR, however, was not affected by low-calcium conditions, indicating other pathways in addition to DDR signaling can likely lead to direct Mk specification from HSCs. Collectively, our work establishes that preventing direct Mk commitment in HSCs, either by preventing uracil incorporation or by culture in low-calcium conditions, enhances HSC maintenance, thereby establishing that the propensity to directly engage the Mk pathway is a barrier to HSC maintenance. These findings will have important implications for future efforts at manipulating HSCs in vitro and at in vivo hematopoietic recovery after insults such as irradiation, chemotherapy, and inflammation. Furthermore, two arguments support the notion that this work may have uncovered an important tumor suppressor mechanism. First, the folate cycle, which provides thymidine and prevents uracil misincorporation, is upregulated in most cancers and targeted by several drugs, while folate deficiency is not oncogenic. This suggests that limiting the supply of thymidine in HSCs prevents inadvertent expansion and malignant transformation. Second, our findings indicate that DNA-damaged HSCs, in part through uracil misincorporation, rapidly generate a lineage essential to immediate organismal survival, thus removing potentially mutated cells from the HSC pool to avoid malignant transformation. Finally, we also attempted to study the in vivo relevance of calcium regulation of HSCs. HSCs reside in the BM, and as bone is the main calcium buffering in the body. We therefore initiated studies to investigate whether changes in bone turnover, potentially mediated by changes in microenvironmental calcium concentration, affect HSCs function. Although difficult to directly correlated with calcium conditions in vitro, our findings indicate that both increased and decreased bone turnover do affect HSC function in vivo. Interestingly, bone turnover differentially affects HSCs with mutation in Tet2. These observations may have clinical significance as recent studies revealed that premature menopause, which is associated with increased bone turnover, accelerates the development of clonal hematopoiesis, a condition caused among others by mutation in Tet2.

No description available.

No description available.

INTRODUÇÃO: O objetivo deste estudo foi de avaliar a eficácia de um centrifugado osteogênico de medula óssea para estimular a consolidação de osteotomias da fíbula em coelhos. MÉTODOS: Este estudo experimental envolveu a utilização de dez coelhos machos adultos da raça Nova Zelândia albino. Realizou-se uma osteotomia transversa médio-diafisária da fíbula direita, seguida da adição local de uma esponja de colágeno absorvível embebida em um centrifugado osteogênico, obtido pela centrifugação de aspirado de medula óssea do osso ilíaco ipsilateral. A fíbula esquerda foi utilizada como controle, sendo feita a mesma osteotomia, porém neste caso adicionando-se somente a esponja de colágeno absorvível. O centrifugado de medula óssea elaborado em laboratório foi submetido à contagem do número de células nucleadas e a teste de viabilidade celular antes de ser administrada no local das osteotomias. Após quatro semanas os animais foram sacrificados para estudo dos calos ósseos formados. Os critérios de avaliação foram a mensuração da densidade mineral utilizando-se a densitometria óssea com DEXA, do volume do calo com tomografia computadorizada multi-slice e dos tecidos formados por meio de histomorfometria. RESULTADOS: O método utilizado para a centrifugação dos aspirados de medula óssea resultou em uma concentração média de células nucleadas três vezes maior que o número destas células nos aspirados originais, sem destruição celular significativa. A utilização deste centrifugado osteogênico resultou em um aumento médio na densidade mineral óssea dos calos de 40,3% e da quantidade relativa de tecido ósseo de 9,4%, sem aumento significativo nas quantidades relativas de cartilagem ou fibrose. Não houve aumento significativo no volume dos calos ósseos. CONCLUSÃO: A administração de centrifugado osteogênico de medula óssea utilizado neste estudo favoreceu a consolidação óssea de osteotomias experimentais em coelhos, observando-se uma melhora qualitativa do calo ósseo. / INTRODUTION: The purpose of this study was to evaluate the efficacy of a centrifuged osteogenic bone-marrow aspirate to stimulate rabbit fibular osteotomies healing. METHOD: Ten white New Zeeland rabbits were used. A transverse middle-diaphysis fibular osteotomy was performed at the right fibula, where a collagen absorbable sponge embedded in the osteogenic centrifuged bone marrow aspirate was inserted. The left fibula was used as the control group, where the collagen absorbable sponge was inserted without the osteogenic centrifuged aspirate. The centrifuged bone-marrow aspirate was arranged at the laboratory and submitted to nuclear cell count and cell viability test. The rabbits were killed at four weeks after surgery to evaluate bone callus formation. The results analysis was performed with DEXA bone densitometry to evaluate callus mineral mass, multislice computer tomography to evaluate callus volume and histomorphometry to evaluate the relative rate of tissue formation. RESULTS: The bone-marrow centrifugation technique increased the number of nucleated cells by three compared with the number of that cells in the original bone-marrow aspirates, without significant nucleated cell dead. The apply of centrifuged osteogenic bone-marrow aspirate resulted in an increased callus bone mineral mass by 40,3%, and increased relative rate of bone tissue formation by 9,4%, without increase the relative rate of cartilage or fibrous tissue. There was not increased callus volume. CONCLUSION: This study shows that the centrifuged osteogenic bone-marrow aspirate was able to improve the healing of experimental fibular osteotomies in rabbits by qualitative improve of bone callus.

A terapia celular representa uma abordagem promissora para o tratamento de cardiopatia isquêmica, porém aspectos-chave dessa estratégia permanecem incertos. Neste trabalho avaliamos a eficiência da retenção cardíaca de células da medula óssea marcadas com tecnécio (99m Tc-CMO) transplantadas, de acordo com o tempo após o infarto (1, 2, 3 e 7 dias) e a via de administração dessas células (intravenosa [IV], intraventricular [IC], intracoronariana [ICO] e intramiocárdica [IM]), em ratos submetidos à isquemia-reperfusão cardíaca [I&R]. Após 24 horas, a retenção cardíaca de 99m Tc-CMO foi maior na via IM comparada com a média alcançada pelas demais (6,79% do total injetado vs. 0,53%). O uso de fibrina como veículo para a injeção de células incrementou a retenção em 2.5 vezes (17,12 vs. 6,84%) na via IM. Curiosamente, quando administradas após 7 dias, a retenção de células na via IM alcançou valores próximos dos observados com da matriz de fibrina injetadas 24 h após a I&R (16,55 vs. 17,12%), enquanto que para as demais vias as mudanças foram insignificantes. Nos animais em que as CMO foram administradas por via intramiocárdica 24 horas após a I&R, com ou sem fibrina, observou-se melhora significante do desempenho cardíaco frente ao estresse farmacológico com fenilefrina quando comparados aos controles. Em conjunto, os dados mostram a biodistribuição das células injetadas após a I&R por 4 diferentes vias e 4 intervalos de tempo pós-lesão e indicam que a via IM é a que produz maior retenção cardíaca. O uso do biopolímero de fibrina aumenta a retenção das células e a eficácia deste efeito sobre a função cardíaca e mortalidade dos animais em longo prazo, além de 30 dias pós I&R, merecerá ser investigada no futuro. / Cell therapy represents a promising approach for ischemic cardiac disease, but key aspects of this strategy remain unclear. We examined the effects of timing and route of administration of bone marrow cells (BMCs) after myocardial ischemia/reperfusion injury (I&R). 99mTc-labeled BMCs were injected by 4 different routes: intravenous (IV), left ventricular cavity (LV), left ventricular cavity with temporal aorta occlusion (LV+) and intramyocardial (IM). The injections were performed 1, 2, 3, or 7 days after infarction. Cardiac retention was higher following the IM route compared to the average values obtained by all other routes (6.79% of the total radioactivity injected vs. 0.53%). Use of a fibrin biopolymer as vehicle during IM injection led to a 2.5-fold increase in cardiac cell accumulation (17.12 vs. 6.84%). Interestingly, the retention of cells administered with culture medium at day 7 post-MI by the IM route was similar to that observed when cells were injected 24 h post-IM using fibrin (16.55 vs 17.12%), whereas no significant changes were observed for the other routes. Cell therapy 24 hs post MI by IM injection, with or without fibrin, resulted in comparable improvement in cardiac function under pharmacological stress compared to control animals. Together, we provide evidence for the biodistribution of 99mTc-labeled BMCs injected post MI by 4 different routes and times post-injury, which shows that the IM rout is the most effective for cardiac cell retention. The use of a fibrin biopolymer further increased cardiac cell retention and its potential long term benefits, beyond 30, on reducing mortality and improving cardiac function deserve to be explored in the future.

INTRODUÇÃO: O objetivo deste estudo foi de avaliar a eficácia de um centrifugado osteogênico de medula óssea para estimular a consolidação de osteotomias da fíbula em coelhos. MÉTODOS: Este estudo experimental envolveu a utilização de dez coelhos machos adultos da raça Nova Zelândia albino. Realizou-se uma osteotomia transversa médio-diafisária da fíbula direita, seguida da adição local de uma esponja de colágeno absorvível embebida em um centrifugado osteogênico, obtido pela centrifugação de aspirado de medula óssea do osso ilíaco ipsilateral. A fíbula esquerda foi utilizada como controle, sendo feita a mesma osteotomia, porém neste caso adicionando-se somente a esponja de colágeno absorvível. O centrifugado de medula óssea elaborado em laboratório foi submetido à contagem do número de células nucleadas e a teste de viabilidade celular antes de ser administrada no local das osteotomias. Após quatro semanas os animais foram sacrificados para estudo dos calos ósseos formados. Os critérios de avaliação foram a mensuração da densidade mineral utilizando-se a densitometria óssea com DEXA, do volume do calo com tomografia computadorizada multi-slice e dos tecidos formados por meio de histomorfometria. RESULTADOS: O método utilizado para a centrifugação dos aspirados de medula óssea resultou em uma concentração média de células nucleadas três vezes maior que o número destas células nos aspirados originais, sem destruição celular significativa. A utilização deste centrifugado osteogênico resultou em um aumento médio na densidade mineral óssea dos calos de 40,3% e da quantidade relativa de tecido ósseo de 9,4%, sem aumento significativo nas quantidades relativas de cartilagem ou fibrose. Não houve aumento significativo no volume dos calos ósseos. CONCLUSÃO: A administração de centrifugado osteogênico de medula óssea utilizado neste estudo favoreceu a consolidação óssea de osteotomias experimentais em coelhos, observando-se uma melhora qualitativa do calo ósseo. / INTRODUTION: The purpose of this study was to evaluate the efficacy of a centrifuged osteogenic bone-marrow aspirate to stimulate rabbit fibular osteotomies healing. METHOD: Ten white New Zeeland rabbits were used. A transverse middle-diaphysis fibular osteotomy was performed at the right fibula, where a collagen absorbable sponge embedded in the osteogenic centrifuged bone marrow aspirate was inserted. The left fibula was used as the control group, where the collagen absorbable sponge was inserted without the osteogenic centrifuged aspirate. The centrifuged bone-marrow aspirate was arranged at the laboratory and submitted to nuclear cell count and cell viability test. The rabbits were killed at four weeks after surgery to evaluate bone callus formation. The results analysis was performed with DEXA bone densitometry to evaluate callus mineral mass, multislice computer tomography to evaluate callus volume and histomorphometry to evaluate the relative rate of tissue formation. RESULTS: The bone-marrow centrifugation technique increased the number of nucleated cells by three compared with the number of that cells in the original bone-marrow aspirates, without significant nucleated cell dead. The apply of centrifuged osteogenic bone-marrow aspirate resulted in an increased callus bone mineral mass by 40,3%, and increased relative rate of bone tissue formation by 9,4%, without increase the relative rate of cartilage or fibrous tissue. There was not increased callus volume. CONCLUSION: This study shows that the centrifuged osteogenic bone-marrow aspirate was able to improve the healing of experimental fibular osteotomies in rabbits by qualitative improve of bone callus.

Peripheral vascular diseases such as peripheral artery disease (PAD) and critical limb ischemia (CLI) are growing at an ever-increasing rate in the Western world due to an aging population and the incidence of type II diabetes. A growing economic burden continues because these diseases are common indicators of future heart attack or stroke. Common therapies are generally limited to pharmacologic agents or endovascular therapies which have had mixed results still ending in necrosis or limb loss. Therapeutic angiogenic strategies have become welcome options for patients suffering from PAD due to the restoration of blood flow in the extremities. Capillary sprouting and a return to normoxic tissue states are also demonstrated by the use of angiogenic cytokines in conjunction with bone marrow cell populations. To this point, it has been determined that spatial and temporal controlled release of growth factors from vehicles provides a greater therapeutic and angiogenic effect than growth factors delivered intramuscularly, intravenously, or intraarterialy due to rapid metabolization of the cytokine, and non-targeted release. Furthermore, bone marrow cells have been implicated to enhance angiogenesis in numerous ischemic diseases due to their ability to secrete angiogenic cytokines and their numerous cell fractions present which are implicated to promote mature vessel formation. Use of angiogenic peptides, in conjunction with bone marrow cells, has been hypothesized in EPC mobilization from the periphery and marrow tissues to facilitate neovessel formation. For this purpose, controlled release of angiogenic peptides basic fibroblast growth factor (FGF-2) and granulocyte-colony stimulating factor (G-CSF) was performed using tunable ionic gelatin hydrogels or fibrin scaffolds with ionic albumin microspheres. The proliferation of endothelial cell culture was determined to have an enhanced effect based on altering concentrations of growth factors and method of release: co-delivery versus sequential. Scaffolds with these angiogenic peptides were implanted in young balb/c mice that underwent unilateral hindlimb ischemia by ligation and excision of the femoral artery. Endpoints for hindlimb reperfusion and angiogenesis were determined by Laser Doppler Perfusion Imaging and immunohistochemical staining for capillaries (CD-31) and smooth muscle cells (alpha-SMA). In addition to controlled release of angiogenic peptides, further studies combined the use of a fibrin co-delivery scaffold with FGF-2 and G-CSF with bone marrow stem cell transplantation to enhance vessel formation following CLI. Endpoints also included lipophilic vascular painting to evaluate the extent of angiogenesis and arteriogenesis in an ischemic hindlimb. Tissue engineering strategies utilizing bone marrow cells and angiogenic peptides demonstrate improved hindlimb blood flow compared to BM cells or cytokines alone, as well as enhanced angiogenesis based on immunohistochemical staining and vessel densities.