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1.
Proc Natl Acad Sci U S A ; 117(32): 19033-19044, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32709748

RESUMO

Therapeutic factors secreted by mesenchymal stem cells (MSCs) promote angiogenesis in vivo. However, delivery of MSCs in the absence of a cytoprotective environment offers limited efficacy due to low cell retention, poor graft survival, and the nonmaintenance of a physiologically relevant dose of growth factors at the injury site. The delivery of stem cells on an extracellular matrix (ECM)-based platform alters cell behavior, including migration, proliferation, and paracrine activity, which are essential for angiogenesis. We demonstrate the biophysical and biochemical effects of preconditioning human MSCs (hMSCs) for 96 h on a three-dimensional (3D) ECM-based microgel platform. By altering the macromolecular concentration surrounding cells in the microgels, the proangiogenic phenotype of hMSCs can be tuned in a controlled manner through cell-driven changes in extracellular stiffness and "outside-in" integrin signaling. The softest microgels were tested at a low cell dose (5 × 104 cells) in a preclinical hindlimb ischemia model showing accelerated formation of new blood vessels with a reduced inflammatory response impeding progression of tissue damage. Molecular analysis revealed that several key mediators of angiogenesis were up-regulated in the low-cell-dose microgel group, providing a mechanistic insight of pathways modulated in vivo. Our research adds to current knowledge in cell-encapsulation strategies by highlighting the importance of preconditioning or priming the capacity of biomaterials through cell-material interactions. Obtaining therapeutic efficacy at a low cell dose in the microgel platform is a promising clinical route that would aid faster tissue repair and reperfusion in "no-option" patients suffering from peripheral arterial diseases, such as critical limb ischemia (CLI).


Assuntos
Células-Tronco Mesenquimais/química , Células-Tronco Mesenquimais/citologia , Microgéis/química , Neovascularização Fisiológica , Animais , Proliferação de Células , Células Imobilizadas/química , Células Imobilizadas/citologia , Células Imobilizadas/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Membro Posterior/irrigação sanguínea , Membro Posterior/metabolismo , Membro Posterior/cirurgia , Humanos , Integrinas/genética , Integrinas/metabolismo , Isquemia/fisiopatologia , Isquemia/cirurgia , Isquemia/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Nus
2.
PLoS One ; 15(6): e0235362, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32584895

RESUMO

OBJECTIVE: Cardiovascular disease is a leading cause of death worldwide. Obesity-related metabolic disorders including dyslipidemia cause impaired collateralization under ischemic conditions, thereby resulting in exacerbated cardiovascular dysfunction. Pemafibrate is a novel selective PPARα modulator, which has been reported to improve atherogenic dyslipidemia, in particular, hypertriglyceridemia and low HDL-cholesterol. Here, we investigated whether pemafibrate modulates the revascularization process in a mouse model of hindlimb ischemia. METHODS AND RESULTS: Male wild-type (WT) mice were randomly assigned to two groups, normal diet or pemafibrate admixture diet from the ages of 6 weeks. After 4 weeks, mice were subjected to unilateral hindlimb surgery to remove the left femoral artery and vein. Pemafibrate treatment enhanced blood flow recovery and capillary formation in ischemic limbs of mice, which was accompanied by enhanced phosphorylation of endothelial nitric oxide synthase (eNOS). Treatment of cultured endothelial cells with pemafibrate resulted in increased network formation and migratory activity, which were blocked by pretreatment with the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME). Pemafibrate treatment also increased plasma levels of the PPARα-regulated gene, fibroblast growth factor (FGF) 21 in WT mice. Systemic administration of adenoviral vectors expressing FGF21 (Ad-FGF21) to WT mice enhanced blood flow recovery, capillary density and eNOS phosphorylation in ischemic limbs. Treatment of cultured endothelial cells with FGF21 protein led to increases in endothelial cell network formation and migration, which were canceled by pretreatment with L-NAME. Furthermore, administration of pemafibrate or Ad-FGF21 had no effects on blood flow in ischemic limbs in eNOS-deficient mice. CONCLUSION: These data suggest that pemafibrate can promote revascularization in response to ischemia, at least in part, through direct and FGF21-mediated modulation of endothelial cell function. Thus, pemafibrate could be a potentially beneficial drug for ischemic vascular disease.


Assuntos
Benzoxazóis/farmacologia , Butiratos/farmacologia , Isquemia/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/sangue , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Membro Posterior/irrigação sanguínea , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Knockout , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , PPAR alfa/química , PPAR alfa/metabolismo , Fosforilação/efeitos dos fármacos
3.
Am J Physiol Regul Integr Comp Physiol ; 319(1): R1-R10, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32348680

RESUMO

Dynamic exercise elicits robust increases in sympathetic activity in part due to muscle metaboreflex activation (MMA), a pressor response triggered by activation of skeletal muscle afferents. MMA during dynamic exercise increases arterial pressure by increasing cardiac output via increases in heart rate, ventricular contractility, and central blood volume mobilization. In heart failure, ventricular function is compromised, and MMA elicits peripheral vasoconstriction. Ventricular-vascular coupling reflects the efficiency of energy transfer from the left ventricle to the systemic circulation and is calculated as the ratio of effective arterial elastance (Ea) to left ventricular maximal elastance (Emax). The effect of MMA on Ea in normal subjects is unknown. Furthermore, whether muscle metaboreflex control of Ea is altered in heart failure has not been investigated. We utilized two previously published methods of evaluating Ea [end-systolic pressure/stroke volume (EaPV)] and [heart rate × vascular resistance (EaZ)] during rest, mild treadmill exercise, and MMA (induced via partial reductions in hindlimb blood flow imposed during exercise) in chronically instrumented conscious canines before and after induction of heart failure via rapid ventricular pacing. In healthy animals, MMA elicits significant increases in effective arterial elastance and stroke work that likely maintains ventricular-vascular coupling. In heart failure, Ea is high, and MMA-induced increases are exaggerated, which further exacerbates the already uncoupled ventricular-vascular relationship, which likely contributes to the impaired ability to raise stroke work and cardiac output during exercise in heart failure.


Assuntos
Artérias/fisiopatologia , Insuficiência Cardíaca/fisiopatologia , Músculo Esquelético/metabolismo , Condicionamento Físico Animal , Animais , Artérias/inervação , Cães , Elasticidade , Feminino , Frequência Cardíaca , Membro Posterior/irrigação sanguínea , Masculino , Músculo Esquelético/inervação , Neurônios Aferentes , Reflexo/fisiologia , Volume Sistólico , Resistência Vascular
4.
Can J Physiol Pharmacol ; 98(4): 228-235, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32207632

RESUMO

To test if magnitudes of the beneficial actions of CO2 water bath therapy on blood flow and vascular density are dependent upon temperature, ischemia in the hind limb of rats was induced by occluding the left femoral artery for 2 weeks and the animals were exposed to water bath therapy with or without CO2 at 34 or 41 °C for 4 weeks (20 min treatment each day for 5 days/week). CO2 water bath therapy at 34 °C increased peak, minimal, and mean blood flow by 190%-600% in the ischemic limb. On the other hand, CO2 water bath treatment at 41 °C increased these parameters of blood flow by 37%, 55%, and 41%, respectively, in the ischemic limb. The small blood vessel count, an index of vascular density, in the ischemic limb was increased by CO2 water bath therapy at 34 and 41 °C by 32% and 122%, respectively. No changes in the ischemic animals by CO2 water bath therapy at 34 or 41 °C were observed in the heart rate, R-R interval, and plasma lipid or glucose levels. These data indicate that the beneficial effect of CO2 water bath therapy at 34 °C on blood flow in the ischemic muscle is greater whereas that on vascular density is smaller than changes in these parameters at 41 °C.


Assuntos
Dióxido de Carbono/farmacologia , Membro Posterior/irrigação sanguínea , Membro Posterior/efeitos dos fármacos , Isquemia/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Fluxo Sanguíneo Regional/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Artéria Femoral/efeitos dos fármacos , Hemodinâmica/efeitos dos fármacos , Masculino , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/efeitos dos fármacos , Ratos , Temperatura , Água
5.
Nat Commun ; 11(1): 615, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001693

RESUMO

Angiogenesis induction into damaged sites has long been an unresolved issue. Local treatment with pro-angiogenic molecules has been the most common approach. However, this approach has critical side effects including inflammatory coupling, tumorous vascular activation, and off-target circulation. Here, the concept that a structure can guide desirable biological function is applied to physically engineer three-dimensional channel networks in implant sites, without any therapeutic treatment. Microchannel networks are generated in a gelatin hydrogel to overcome the diffusion limit of nutrients and oxygen three-dimensionally. Hydrogel implantation in mouse and porcine models of hindlimb ischemia rescues severely damaged tissues by the ingrowth of neighboring host vessels with microchannel perfusion. This effect is guided by microchannel size-specific regenerative macrophage polarization with the consequent functional recovery of endothelial cells. Multiple-site implantation reveals hypoxia and neighboring vessels as major causative factors of the beneficial function. This technique may contribute to the development of therapeutics for hypoxia/inflammatory-related diseases.


Assuntos
Indutores da Angiogênese/efeitos adversos , Gelatina/química , Gelatina/farmacologia , Hidrogéis/química , Hidrogéis/farmacologia , Isquemia/terapia , Animais , Modelos Animais de Doenças , Células Endoteliais/patologia , Desenho de Equipamento , Feminino , Membro Posterior/irrigação sanguínea , Membro Posterior/diagnóstico por imagem , Membro Posterior/patologia , Hidrogéis/uso terapêutico , Hipóxia , Isquemia/diagnóstico por imagem , Isquemia/patologia , Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neovascularização Fisiológica/fisiologia , Doenças Vasculares Periféricas/patologia , Doenças Vasculares Periféricas/terapia , Próteses e Implantes , Suínos , Cicatrização
6.
J Surg Res ; 250: 119-124, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32044508

RESUMO

BACKGROUND: Vascularized composite allograft has emerged as a reconstructive option for patients who have suffered severe tissue loss. Animal models are critical for understanding the unique mechanisms of rejection in vascularized composite allograft. We present a functional mouse model of orthotopic hind limb transplantation using end-to-side anastomoses of the donor aorta and inferior vena cava to the respective recipient vessels. To the best of our knowledge, this approach has not been reported in the scientific literature. MATERIALS AND METHODS: A single surgeon performed all transplants (J.W.). A total of 13 syngeneic and 10 fully mismatched allogeneic transplants were performed without immunosuppression. Skin samples from the grafts were collected at the time of euthanasia. RESULTS: Five syngeneic mice survived for more than 90 d after transplant. All allografts displayed clinical and histologic signs of acute rejection such as a rash at the time of graft excision. The overall technical success rate of all transplants in this study was 74% (17 of 23). CONCLUSIONS: We demonstrate the feasibility of end-to-side anastomoses of the donor aorta and inferior vena cava with functional recovery of the transplant in a mouse model of orthotopic hind limb transplantation.


Assuntos
Aloenxertos Compostos/transplante , Rejeição de Enxerto/prevenção & controle , Membro Posterior/transplante , Alotransplante de Tecidos Compostos Vascularizados/métodos , Anastomose Cirúrgica/efeitos adversos , Anastomose Cirúrgica/métodos , Animais , Aorta/cirurgia , Modelos Animais de Doenças , Estudos de Viabilidade , Rejeição de Enxerto/etiologia , Sobrevivência de Enxerto , Membro Posterior/irrigação sanguínea , Humanos , Masculino , Camundongos , Transplante Homólogo/efeitos adversos , Transplante Homólogo/métodos , Alotransplante de Tecidos Compostos Vascularizados/efeitos adversos , Veia Cava Inferior/cirurgia
7.
Pain Physician ; 23(1): E51-E60, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32013288

RESUMO

BACKGROUND: The success rate for the production of animal models of chronic postischemia pain (CPIP) using an O-ring has yet to be improved in the study of complex regional pain syndrome-type I (CRPS-I), and producing a CPIP model is challenging, especially for mice. OBJECTIVES: We devised a new CPIP model with a higher success rate that induces ischemia for 3 hours by tying the hind limbs of mice with a rubber band, followed by reperfusion. STUDY DESIGN: A randomized, controlled animal trial. METHODS: Twenty-two male C57BL/6 mice were divided into a sham (n = 6), a ring (n = 8), and a tie group (n = 8). Anesthesia was induced using isoflurane. A precut O-ring was mounted on the upper left ankle in the sham group. A tight-fitting O-ring and a push-pull gauge manometer were mounted at the same location in the ring and tie groups, respectively. Reperfusion was induced 3 hours later. The thickness and circumference of the hind paws were measured before ischemia induction. Measurements were repeated 10 days after reperfusion. Mechanical allodynia was measured with a von Frey filament until 12 weeks after reperfusion. RESULTS: The new tie model required 5 additional days until the onset of allodynia compared with the existing CPIP O-ring model. However, the successful induction rate of CPIP was higher in the tie group than in the ring group, and allodynia was maintained for over 30 days in the tie group. The ring and tie groups exhibited significantly high levels of tumor necrosis factor-alpha than those in the sham group. LIMITATIONS: First, we did not evaluate hyperalgesia, cold or heat allodynia. Second, we did not measure blood levels of inflammatory or antiinflammatory cytokines, and research on oxidative stress biomarkers such as isoprostane, 8-hydroxy-2'-deoxyguanosine (a marker of DNA oxidative damage), and malondialdehyde was not performed. CONCLUSIONS: The new CPIP tie model has a higher rate of successful induction than existing O-ring models for mice, with longer duration of mechanical allodynia. The model may reduce the number of animals sacrificed in CRPS-I research and could be useful for studying long-term effects of drugs. KEY WORDS: CPIP, mouse, O-ring, rubber band, reperfusion, allodynia, hyperalgesia.


Assuntos
Síndromes da Dor Regional Complexa/patologia , Síndromes da Dor Regional Complexa/fisiopatologia , Modelos Animais de Doenças , Membro Posterior/irrigação sanguínea , Isquemia/patologia , Isquemia/fisiopatologia , Animais , Dor Crônica/patologia , Dor Crônica/fisiopatologia , Constrição Patológica/sangue , Constrição Patológica/patologia , Hiperalgesia/patologia , Hiperalgesia/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Distribuição Aleatória
8.
Arterioscler Thromb Vasc Biol ; 40(4): e105-e113, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32075417

RESUMO

OBJECTIVE: Vascular progenitor cells (VPCs), which are able to differentiate into both endothelial cells and smooth muscle cells, have the potential for treatment of ischemic diseases. Generated by pluripotent stem cells, VPCs carry the risk of tumorigenicity in clinical application. This issue could be resolved by direct lineage conversion, the induction of functional cells from another lineage by using only lineage-restricted transcription factors. Here, we show that induced VPCs (iVPCs) can be generated from fibroblasts by ETS (E-twenty six) transcription factors, Etv2 and Fli1. Approach and Results: Mouse fibroblasts were infected with lentivirus encoding Etv2 and Fli1. Cell colonies appeared in Fli1- and Etv2/Fli1-infected groups and were mechanically picked. The identity of cell colonies was confirmed by proliferation assay and reverse-transcription polymerase chain reaction with vascular markers. Etv2/Fli1- infected cell colonies were sorted by CD144 (also known as CDH5, VE-cadherin). We defined that CD144-positive iVPCs maintained its own population and expanded stably at multiple passages. iVPCs could differentiate into functional endothelial cells and smooth muscle cells by a defined medium. The functionalities of iVPC-derived endothelial cells and smooth muscle cells were confirmed by analyzing LDL (low-density lipoprotein) uptake, carbachol-induced contraction, and tube formation in vitro. Transplantation of iVPCs into the ischemic hindlimb model enhanced blood flow without tumor formation in vivo. Human iVPCs were generated by human ETS transcription factors ETV2 and FLI1. CONCLUSIONS: We demonstrate that ischemic disease curable iVPCs, which have self-renewal and bipotency, can be generated from mouse fibroblasts by enforced ETS family transcription factors, Etv2 and Fli1 expression. Our simple strategy opens insights into stem cell-based ischemic disease therapy.


Assuntos
Fibroblastos/citologia , Isquemia/fisiopatologia , Proteína Proto-Oncogênica c-fli-1/fisiologia , Células-Tronco/fisiologia , Fatores de Transcrição/fisiologia , Animais , Antígenos CD , Caderinas , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Modelos Animais de Doenças , Células Endoteliais/citologia , Membro Posterior/irrigação sanguínea , Isquemia/terapia , Miócitos de Músculo Liso/citologia , Transplante de Células-Tronco , Células-Tronco/imunologia
9.
J Surg Res ; 249: 168-179, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31986359

RESUMO

BACKGROUND: Development of collateral vasculature is key in compensating for arterial occlusions in patients with peripheral artery disease (PAD). We aimed to examine the development of collateral pathways after ligation of native vessels in a porcine model of PAD. METHODS: Right hindlimb ischemia was induced in domestic swine (n = 11) using two versions of arterial ligation. Version 1 (n = 6) consisted of ligation with division of the right external iliac, profunda femoral, and superficial femoral arteries. Version 2 (n = 5) consisted of the ligation of version 1 with additional ligation with division of the right internal iliac artery. Development of collateral pathways was evaluated with standard angiography before arterial ligation and at termination (30 days later). Relative luminal diameter of the arteries supplying the ischemic right hind limb were determined by two-dimensional angiography. RESULTS: The dominant collateral pathway that developed after version 1 ligation connected the right internal iliac artery to the right profunda femoral and then to the right superficial femoral and popliteal artery. Mean luminal diameter of the right internal iliac artery at termination increased by 38% compared with baseline. Two codominant collateral pathways developed in version 2 ligation: (i) from the left profunda femoral artery to the reconstituted right profunda femoral artery and (ii) from the common internal iliac trunk and the left internal iliac artery to the reconstituted right internal iliac artery, which then supplied the right profunda femoral and then the right superficial femoral and popliteal artery. The mean diameter of the left profunda and the left internal iliac artery increased at termination by 26% and 21%, respectively (P < 0.05). CONCLUSIONS: Two versions of hindlimb ischemia induction (right ilio-femoral artery ligation with and without right internal iliac artery ligation) in swine produced differing collateral pathways, along with changes to the diameter of the inflow vessels (i.e., arteriogenesis).


Assuntos
Circulação Colateral/fisiologia , Isquemia/fisiopatologia , Neovascularização Fisiológica/fisiologia , Doença Arterial Periférica/fisiopatologia , Angiografia , Animais , Modelos Animais de Doenças , Artéria Femoral/diagnóstico por imagem , Artéria Femoral/cirurgia , Membro Posterior/irrigação sanguínea , Humanos , Artéria Ilíaca/diagnóstico por imagem , Artéria Ilíaca/cirurgia , Isquemia/diagnóstico por imagem , Isquemia/etiologia , Ligadura/efeitos adversos , Masculino , Doença Arterial Periférica/diagnóstico por imagem , Doença Arterial Periférica/etiologia , Fluxo Sanguíneo Regional/fisiologia , Sus scrofa
10.
FASEB J ; 34(1): 1231-1246, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914695

RESUMO

Endothelial progenitor cells (EPCs) promote neovascularization and tissue repair by migrating to vascular injury sites; therefore, factors that enhance EPC homing to damaged tissues are of interest. Here, we provide evidence of the prominent role of the Netrin-4 (NTN4)-Unc-5 Netrin receptor B (UNC5B) axis in EPC-specific promotion of ischemic neovascularization. Our results showed that NTN4 promoted the proliferation, chemotactic migration, and paracrine effects of small EPCs (SEPCs) and significantly increased the incorporation of large EPCs (LEPCs) into tubule networks. Additionally, NTN4 prominently augmented neovascularization in mice with hindlimb ischemia by increasing the homing of exogenously transplanted EPCs to the ischemic limb and incorporating EPCs into vessels. Moreover, silencing of UNC5B, an NTN4 receptor, abrogated the NTN4-induced cellular activities of SEPCs in vitro and blood-flow recovery and neovascularization in vivo in ischemic muscle by reducing EPC homing and incorporation. These findings suggest NTN4 as an EPC-based therapy for treating angiogenesis-dependent diseases.


Assuntos
Células Progenitoras Endoteliais/metabolismo , Isquemia/metabolismo , Músculo Esquelético/metabolismo , Neovascularização Patológica/metabolismo , Receptores de Netrina/metabolismo , Netrinas/metabolismo , Animais , Células Progenitoras Endoteliais/patologia , Células Progenitoras Endoteliais/transplante , Inativação Gênica , Xenoenxertos , Membro Posterior/irrigação sanguínea , Humanos , Isquemia/genética , Isquemia/patologia , Isquemia/terapia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/patologia , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Neovascularização Patológica/terapia , Receptores de Netrina/genética , Netrinas/genética
11.
J Plast Surg Hand Surg ; 54(1): 59-65, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31702408

RESUMO

The aim of this study was to verify the possibility of preparation and effectiveness of the use of blood plasma containing an effector of ischemic tolerance activated by applying two sublethal stresses to a donor. As sublethal stresses, two periods of 20-minute hindlimb ischemia were used with a two-day interval between them. Active plasma was isolated six hours after the second hindlimb ischemia. The effectiveness of active plasma as well as remote postconditioning was tested after three hours of tourniquet-induced ischemia on the gastrocnemius muscle. The wet/dry ratio of gastrocnemius muscle (degree of tissue oedema), nitroblue tetrazolium reduction (tissue necrosis), and CatWalk test (hind limb functionality) were evaluated 24 h after the end of ischemia. Three hours of ischemia increased muscle oedema and necrosis in comparison to control by 26.72% (p < 0.001) and 41.58% (p < 0.001) respectively. Remote ischemic postconditioning as well as injection of conditioned blood plasma significantly prevented these changes, even when they were applied one or three hours after the end of ischemia. Equally effective double-conditioned plasma appears to have better prospects in life-threatening situations such as stroke and myocardial infarction.


Assuntos
Membro Posterior/irrigação sanguínea , Pós-Condicionamento Isquêmico/métodos , Músculo Esquelético/irrigação sanguínea , Plasma , Traumatismo por Reperfusão/prevenção & controle , Animais , Modelos Animais de Doenças , Edema/patologia , Músculo Esquelético/patologia , Necrose , Distribuição Aleatória , Ratos Wistar
12.
Arterioscler Thromb Vasc Biol ; 40(1): 239-254, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31665908

RESUMO

OBJECTIVES: Critical hindlimb ischemia is a severe consequence of peripheral artery disease. Surgical treatment does not prevent skeletal muscle impairment or improve long-term patient outcomes. The present study investigates the protective/regenerative potential and the mechanism of action of adipose stem cell-derived extracellular vesicles (ASC-EVs) in a mouse model of hindlimb ischemia. Approach and Results: We demonstrated that ASC-EVs exert a protective effect on muscle damage by acting both on tissue microvessels and muscle cells. The genes involved in muscle regeneration were up-regulated in the ischemic muscles of ASC-EV-treated animals. MyoD expression has also been confirmed in satellite cells. This was followed by a reduction in muscle function impairment in vivo. ASC-EVs drive myoblast proliferation and differentiation in the in vitro ischemia/reoxygenation model. Moreover, ASC-EVs have shown an anti-apoptotic effect both in vitro and in vivo. Transcriptomic analyses have revealed that ASC-EVs carry a variety of pro-angiogenic mRNAs, while proteomic analyses have demonstrated an enrichment of NRG1 (neuregulin 1). A NRG1 blocking antibody used in vivo demonstrated that NRG1 is relevant to ASC-EV-induced muscle protection, vascular growth, and recruitment of inflammatory cells. Finally, bioinformatic analyses on 18 molecules that were commonly detected in ASC-EVs, including mRNAs and proteins, confirmed the enrichment of pathways involved in vascular growth and muscle regeneration/protection. CONCLUSIONS: This study demonstrates that ASC-EVs display pro-angiogenic and skeletal muscle protective properties that are associated with their NRG1/mRNA cargo. We, therefore, propose that ASC-EVs are a useful tool for therapeutic angiogenesis and muscle protection.


Assuntos
Adipócitos/citologia , Vesículas Extracelulares/metabolismo , Membro Posterior/irrigação sanguínea , Isquemia/patologia , Músculo Esquelético/ultraestrutura , Neuregulina-1/metabolismo , Células-Tronco/ultraestrutura , Adipócitos/metabolismo , Animais , Western Blotting , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Vesículas Extracelulares/ultraestrutura , Isquemia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Músculo Esquelético/metabolismo , Proteômica , Células-Tronco/metabolismo
13.
J Ethnopharmacol ; 248: 112330, 2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-31654796

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Seven traditional medicinal plants (including Astragalus membranaceus, Dioscorea hemsleyi, Salvia miltiorrhiza, Scrophularia ningpoensis, Ophiopogon japonicus, Panax ginseng and Fritillariae cirrhosae) and one insect leech (Whitmania pigra Whitman) were combined into BuZangTongLuo formula (BZTLF) under the guidance of traditional Chinese medicine. BZTLF is potentially effective against diabetic vascular complications. AIM OF THE STUDY: Previous studies failed to clarify the molecular mechanism through which BZTLF suppressed diabetic ischemia. In this study, we aimed to explore whether BZTLF treatment could prevent the occurrence of type 2 diabetic (T2D) hindlimb ischemia in mice. Further, we investigated the regulatory effect of BZTLF on angiogenesis-related VEGF signaling pathway and gut microbiota dysfunction in diabetic ischemia mice. MATERIALS AND METHODS: C57BL/6J mice fed with high-fat diet (HFD) received STZ injection and femoral artery ligation to build T2D diabetic hindlimb ischemia model. Mice were gavaged with BZTLF (5 g [raw materials]/kg/d) or with metformin plus atorvastatin for three weeks. Laser doppler imaging system was utilized for the visualization of blood flow. Histochemistry analysis was performed for microvascular vessel staining. Western blot was applied to detect the protein changes of signaling molecules responsible for VEGF pathway. Finally, 16S rDNA gene sequencing was conducted for analysis of gut microbiota structure. RESULTS: BZTLF treatment remarkably restored blood flow and capillary density of diabetic hindlimb ischemia. And the protein changes of VEGF signaling molecules were reversed in BZTLF-treated diabetic ischemia mice, including the decreased VEGF and HIF-1α, and the increased NO, eNOS and p-ERK1/2. The gut microbiota analysis suggests that BZTLF treatment increased the abundances of several beneficial bacteria (Akkermansia, Bifidobacterium and Bacteroides), while decreased the populations of some harmful bacteria(Blautia, Weissella, Escherichia Shigella and Kurthia). By using Spearman's correlation analysis, these changed gut flora were positively/negatively correlated with VEGF signaling pathway or glycometabolic parameters. CONCLUSION: BZTLF displayed beneficial effects on diabetic hindlimb ischemia by reshaping the gut microbiota structure and stunning the VEGF/HIF-1α pathway.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Angiopatias Diabéticas/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Membro Posterior/irrigação sanguínea , Isquemia/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Velocidade do Fluxo Sanguíneo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/microbiologia , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/microbiologia , Diabetes Mellitus Tipo 2/fisiopatologia , Angiopatias Diabéticas/metabolismo , Angiopatias Diabéticas/microbiologia , Angiopatias Diabéticas/fisiopatologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isquemia/metabolismo , Isquemia/microbiologia , Isquemia/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fluxo Sanguíneo Regional , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo
14.
Can J Cardiol ; 35(11): 1546-1556, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31679624

RESUMO

BACKGROUND: Endothelial progenitor cell (EPC) therapy has been suggested as a major breakthrough in the treatment of ischemic diseases. However, the molecular mechanism that underlies EPC functional regulation is still unclear. METHODS: We examined the angiogenic capacity of EPCs in a hindlimb ischemia model of wild-type and ClC-3 knockout mice. RESULTS: Mice lacking of ClC-3 exhibited reduced blood flow recovery and neovascularization in ischemic muscles 7 and 14 days after hind limb ischemia. Moreover, compared with wild-type EPCs, the hindlimb blood reperfusion in mice receiving ClC-3 knockout EPCs was significantly impaired, accompanied by reduced EPC homing and retention. In vitro, EPCs derived from ClC-3 knockout mice displayed impaired migratory, adhesive, and angiogenic activity. CXC chemokine receptor 4 (CXCR4) expression was significantly reduced in EPC from ClC-3 knockout mice compared with wild-type. Moreover, the expression and phosphorylation of Janus kinase 2 (JAK-2), a downstream signalling of CXCR4, was also reduced in ClC-3 knockout EPC, indicating that CXCR4/JAK-2 signalling is dysregulated by ClC-3 deficiency. Consistent with this assumption, the migratory capacity of wild-type EPCs was attenuated by either CXCR4 antagonist AMD3100 or JAK-2 inhibitor AG490. More importantly, the impaired migratory capacity of ClC-3 knockout EPCs was rescued by overexpression of CXCR4. CONCLUSIONS: ClC-3 plays a critical role in the angiogenic capacity of EPCs and EPC-mediated neovascularization of ischemic tissues. Disturbance of CXCR4/JAK-2 signalling may contribute to the functional impairment of ClC-3 deficient EPCs. Thus, ClC-3 may be a potential therapeutic target for modulating neovascularization in ischemic diseases.


Assuntos
Canais de Cloreto/genética , Regulação da Expressão Gênica , Isquemia/metabolismo , Janus Quinase 2/genética , Neovascularização Patológica/metabolismo , Receptores CXCR4/genética , Transplante de Células-Tronco/métodos , Animais , Western Blotting , Células Cultivadas , Canais de Cloreto/biossíntese , Canais de Cloreto/deficiência , Modelos Animais de Doenças , Células Progenitoras Endoteliais/citologia , Células Progenitoras Endoteliais/metabolismo , Membro Posterior/irrigação sanguínea , Isquemia/patologia , Isquemia/terapia , Janus Quinase 2/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Musculares , Neovascularização Patológica/patologia , Neovascularização Patológica/terapia , Receptores CXCR4/biossíntese , Transdução de Sinais
15.
Int J Mol Sci ; 20(18)2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31540534

RESUMO

In recent years, mesenchymal stem cells (MSCs) have emerged as a promising therapeutic modality in regenerative medicine. They hold great promise for treating civilization-wide diseases, including cardiovascular diseases, such as acute myocardial infarction and critical limb ischemia. MSCs isolated from Wharton's jelly (WJ-MSCs) may be utilized in both cell-based therapy and vascular graft engineering to restore vascular function, thereby providing therapeutic benefits for patients. The efficacy of WJ-MSCs lies in their multipotent differentiation ability toward vascular smooth muscle cells, endothelial cells and other cell types, as well as their capacity to secrete various trophic factors, which are potent in promoting angiogenesis, inhibiting apoptosis and modulating immunoreaction. Ischemic limb disease is caused by insufficient nutrient and oxygen supplies resulting from damaged peripheral arteries. The lack of nutrients and oxygen causes severe tissue damage in the limb, thereby resulting in severe morbidities and mortality. The therapeutic effects of the conventional treatments are still not sufficient. Cell transplantations in small animal model (mice) are vital for deciphering the mechanisms of MSCs' action in muscle regeneration. The stimulation of angiogenesis is a promising strategy for the treatment of ischemic limbs, restoring blood supply for the ischemic region. In the present study, we focus on the therapeutic properties of the human WJ-MSCs derived product, Cardio. We investigated the role of CardioCell in promoting angiogenesis and relieving hindlimb ischemia. Our results confirm the healing effect of CardioCell and strongly support the use of the WJ-MSCs in regenerative medicine.


Assuntos
Membro Posterior/irrigação sanguínea , Isquemia/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Geleia de Wharton/citologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Humanos , Masculino , Transplante de Células-Tronco Mesenquimais/métodos , Camundongos , Camundongos SCID , Neovascularização Fisiológica , Regeneração
16.
Nat Commun ; 10(1): 4223, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31530804

RESUMO

Diseases related to impaired blood flow such as peripheral artery disease (PAD) impact nearly 10 million people in the United States alone, yet patients with clinical manifestations of PAD (e.g., claudication and limb ischemia) have limited treatment options. In ischemic tissues, stress kinases such as c-Jun N-terminal kinases (JNKs), are activated. Here, we show that inhibition of the JNK3 (Mapk10) in the neural compartment strikingly potentiates blood flow recovery from mouse hindlimb ischemia. JNK3 deficiency leads to upregulation of growth factors such as Vegfa, Pdgfb, Pgf, Hbegf and Tgfb3 in ischemic muscle by activation of the transcription factors Egr1/Creb1. JNK3 acts through Forkhead box O3 (Foxo3a) to suppress the activity of Egr1/Creb1 transcription regulators in vitro. In JNK3-deficient cells, Foxo3a is suppressed which leads to Egr1/Creb1 activation and upregulation of downstream growth factors. Collectively, these data suggest that the JNK3-Foxo3a-Egr1/Creb1 axis coordinates the vascular remodeling response in peripheral ischemia.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Membro Posterior/irrigação sanguínea , Isquemia/metabolismo , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Neurônios/metabolismo , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Membro Posterior/inervação , Membro Posterior/metabolismo , Humanos , Isquemia/genética , Isquemia/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 10 Ativada por Mitógeno/genética , Músculo Esquelético/metabolismo , Fluxo Sanguíneo Regional , Transdução de Sinais
17.
Can J Physiol Pharmacol ; 97(12): 1193-1203, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31505123

RESUMO

Mammalian target of rapamycin (mTOR) has been recognized with potential immunomodulatory properties playing an important role in various physiopathological processes including ischemia-reperfusion (I/R) injury. I/R injury stimulate reactive oxygen and nitrogen species by activating nicotinamide adenine dinucleotide phosphate oxidase and inducible nitric oxide synthase, respectively. Controversial results have been obtained in different I/R models following localized I/R; however, the precise role of the mTOR signaling pathway remains undefined. The objective of the current study was to evaluate the role of the mTOR in oxidative-nitrosative stress and inflammation in hindlimb I/R-induced injury in target and remote organ injuries. In rats subjected to I/R, an increased expression of ribosomal protein S6 (rpS6), inhibitor κB (IκB)-α, nuclear factor-κB (NF-κB) p65, inducible nitric oxide synthase, cyclooxygenase 2, gp91phox, and levels of tumor necrosis factor α, nitrite, nitrotyrosine, malondialdehyde and the activities of myeloperoxidase and catalase in the tissues and (or) sera were detected. Treatment with rapamycin, a selective inhibitor of mTOR, reversed all the I/R-induced changes as manifested by its anti-inflammatory and antioxidant effects in kidney and gastrocnemius muscle of rats. Collectively, these findings suggest that rapamycin protects against I/R-induced oxidative-nitrosative stress and inflammation leading to organ injuries via suppression of mTOR/IκB-α/NF-κB signaling pathway.


Assuntos
Membro Posterior/irrigação sanguínea , Estresse Nitrosativo/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Traumatismo por Reperfusão/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Biomarcadores/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Inflamação/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Inibidor de NF-kappaB alfa/metabolismo , Ratos , Ratos Wistar , Proteína S6 Ribossômica/metabolismo , Fator de Transcrição RelA/metabolismo
18.
PLoS One ; 14(8): e0220898, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31419236

RESUMO

Batroxobin, isolated from Bothrops moojeni, is a defibrinogenating agent used as a thrombin-like serine protease against fibrinogen for improving microcirculation. Here, we investigated whether, and if so, how batroxobin restores ischemic tissue injury in terms of anti-inflammatory effects. In an in vitro flow cytometry assay for human neutrophil extracellular traps (NETs), batroxobin (DF-521; Defibrase) inhibited human NETs induced by tumor necrosis factor-α (TNF-α) in the presence of human fibrinogen. Next, the effect of batroxobin was investigated by immunohistochemistry of the anterior tibial muscle (ATM) in an ischemic hindlimb model using C57BL/6J mice intraperitoneally injected with DF-521 versus the saline control. NETs and fibrinogen deposition in the ischemic ATM decreased in DF-521-treated mice on day 2 after ischemia. Meanwhile, reverse transcription-quantitative PCR assay of the ischemic ATM unveiled continuous downregulation in the expression of the genes; Tnf-α and nitric oxide synthase2 (Nos2) with hypoxia-inducible factor-1α (Hif-1α) and vascular endothelial growth factor-a (Vegf-a) from day 3 to day 7, but the upregulation of arginase-1 (Arg-1) and placental growth factor (Plgf) with myogenin (Myog) on day 7. Daily intraperitoneal DF-521 injection for the initial 7 days into mice with ischemic hindlimbs promoted angiogenesis and arteriogenesis on day 14. Moreover, DF-521 injection accelerated myofiber maturation after day 14. Laser doppler imaging analysis revealed that blood perfusion in DF-521-injected mice significantly improved on day 14 versus the saline control. Thus, DF-521 improves microcirculation by protecting NETs with tissue defibrinogenation, thereby protecting against severe ischemic tissue injury and accelerating vascular and skeletal muscular regeneration. To our knowledge, batroxobin might be the first clinically applicable NET inhibitor against ischemic diseases.


Assuntos
Batroxobina/uso terapêutico , Armadilhas Extracelulares/efeitos dos fármacos , Fibrinolíticos/uso terapêutico , Isquemia/tratamento farmacológico , Adulto , Animais , Anti-Inflamatórios/uso terapêutico , Células Cultivadas , Modelos Animais de Doenças , Armadilhas Extracelulares/imunologia , Membro Posterior/irrigação sanguínea , Humanos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Isquemia/imunologia , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Cicatrização/efeitos dos fármacos , Adulto Jovem
19.
Opt Lett ; 44(15): 3773-3776, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31368965

RESUMO

Hypoxia, a low tissue oxygenation condition caused by insufficient oxygen supply, leads to potentially irreversible tissue damage, such as brain infarction during stroke. Intravascular oxygenation has long been used by photoacoustic imaging, among other imaging modalities, to study hypoxia. However, intravascular oxygenation describes only the oxygen supply via microcirculation, which does not directly reflect the amount of free oxygen available for metabolism in the interstitial fluid. Therefore, to fully understand hypoxia, it is highly desirable to monitor blood oxygenation as well as tissue oxygenation during the same biological process. In this work, by combining high-resolution photoacoustic microscopy (PAM) and a novel bioreducible N-oxide-based hypoxia-sensitive probe HyP-650, we have demonstrated simultaneous imaging of intravascular oxygenation and tissue hypoxia. We have established detailed chemical, optical, and photoacoustic properties of HyP-650 for hypoxic activation in vitro and in living cells. We have also performed PAM on hindlimb ischemia models and tumor-bearing mice to study the correlation between intravascular oxygenation and tissue oxygenation at various hypoxic levels. We expect that Hyp-650 enhanced photoacoustic imaging will find a variety of applications in brain and cancer research.


Assuntos
Vasos Sanguíneos/diagnóstico por imagem , Vasos Sanguíneos/metabolismo , Oxigênio/metabolismo , Técnicas Fotoacústicas/métodos , Animais , Membro Posterior/irrigação sanguínea , Isquemia/metabolismo , Isquemia/patologia , Camundongos , Microscopia , Hipóxia Tumoral
20.
Int J Mol Sci ; 20(15)2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31362356

RESUMO

Mouse hind limb ischemia is the most common used preclinical model for peripheral arterial disease and critical limb ischemia. This model is used to investigate the mechanisms of neovascularization and to develop new therapeutic agents. The literature shows many variations in the model, including the method of occlusion, the number of occlusions, and the position at which the occlusions are made to induce hind limb ischemia. Furthermore, predefined end points and the histopathological and radiological analysis vary. These differences hamper the correlation of results between different studies. In this review, variations in surgical methods of inducing hind limb ischemia in mice are described, and the consequences of these variations on perfusion restoration and vascular remodeling are discussed. This study aims at providing the reader with a comprehensive overview of the methods so far described, and proposing uniformity in research of hind limb ischemia in a mouse model.


Assuntos
Membro Posterior/irrigação sanguínea , Isquemia/diagnóstico , Isquemia/etiologia , Neovascularização Fisiológica , Complicações Pós-Operatórias , Procedimentos Cirúrgicos Operatórios , Animais , Modelos Animais de Doenças , Membro Posterior/anatomia & histologia , Membro Posterior/patologia , Membro Posterior/cirurgia , Camundongos , Imagem de Perfusão , Fluxo Sanguíneo Regional , Procedimentos Cirúrgicos Operatórios/efeitos adversos , Procedimentos Cirúrgicos Operatórios/métodos , Ultrassonografia Doppler
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