RESUMO
Understanding the mechanisms underlying vascular regeneration in the heart is crucial for developing novel therapeutic strategies for myocardial ischemia. This study investigates the contribution of bone marrow-derived cells to endothelial cell populations in the heart, and their role in cardiac function and coronary circulation following repetitive ischemia (RI). Chimeric rats were created by transplanting BM cells from GFP female rats into irradiated male recipients. After engraftment chimeras were subjected to RI for 17 days. Vascular growth was assessed from recovery of cardiac function and increases in myocardial blood flow during LAD occlusion. After sorting GFP+ BM cells from heart and bone of Control and RI rats, single-cell RNA sequencing was implemented to determine the fate of BM cells. Our in vivo RI model demonstrated an improvement in cardiac function and myocardial blood flow after 17 days of RI with increased capillary density in the rats subjected to RI compared to Controls. Single-cell RNA sequencing of bone marrow cells isolated from rats' hearts identified distinct endothelial cell (EC) subpopulations. These ECs exhibited heterogeneous gene expression profiles and were enriched for markers of capillary, artery, lymphatic, venous, and immune ECs. Furthermore, BM-derived ECs in the RI group showed an angiogenic profile, characterized by upregulated genes associated with blood vessel development and angiogenesis. This study elucidates the heterogeneity of bone marrow-derived endothelial cells in the heart and their response to repetitive ischemia, laying the groundwork for targeting specific subpopulations for therapeutic angiogenesis in myocardial ischemia.
Assuntos
Transplante de Medula Óssea , Modelos Animais de Doenças , Células Endoteliais , Ratos Transgênicos , Animais , Masculino , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Neovascularização Fisiológica , Isquemia Miocárdica/patologia , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/fisiopatologia , Células da Medula Óssea/metabolismo , Circulação Coronária , Miocárdio/patologia , Miocárdio/metabolismo , Ratos , Ratos Sprague-Dawley , TranscriptomaRESUMO
BACKGROUND AND AIMS: Takotsubo syndrome (TTS) is a conundrum without consensus about the cause. In a murine model of coronary microvascular dysfunction (CMD), abnormalities in myocardial perfusion played a key role in the development of TTS. METHODS AND RESULTS: Vascular Kv1.5 channels connect coronary blood flow to myocardial metabolism and their deletion mimics the phenotype of CMD. To determine if TTS is related to CMD, wild-type (WT), Kv1.5-/-, and TgKv1.5-/- (Kv1.5-/- with smooth muscle-specific expression Kv1.5 channels) mice were studied following transaortic constriction (TAC). Measurements of left ventricular (LV) fractional shortening (FS) in base and apex, and myocardial blood flow (MBF) were completed with standard and contrast echocardiography. Ribonucleic Acid deep sequencing was performed on LV apex and base from WT and Kv1.5-/- (control and TAC). Changes in gene expression were confirmed by real-time-polymerase chain reaction. MBF was increased with chromonar or by smooth muscle expression of Kv1.5 channels in the TgKv1.5-/-. TAC-induced systolic apical ballooning in Kv1.5-/-, shown as negative FS (P < 0.05 vs. base), which was not observed in WT, Kv1.5-/- with chromonar, or TgKv1.5-/-. Following TAC in Kv1.5-/-, MBF was lower in LV apex than in base. Increasing MBF with either chromonar or in TgKv1.5-/- normalized perfusion and function between LV apex and base (P = NS). Some genetic changes during TTS were reversed by chromonar, suggesting these were independent of TAC and more related to TTS. CONCLUSION: Abnormalities in flow regulation between the LV apex and base cause TTS. When perfusion is normalized between the two regions, normal ventricular function is restored.
Assuntos
Cardiomiopatia de Takotsubo , Animais , Camundongos , Cromonar , Circulação Coronária/fisiologia , Ecocardiografia , Isquemia Miocárdica , MiocárdioRESUMO
Endothelial dysfunction in diabetes is generally attributed to oxidative stress, but this view is challenged by observations showing antioxidants do not eliminate diabetic vasculopathy. As an alternative to oxidative stress-induced dysfunction, we interrogated if impaired mitochondrial function in endothelial cells is central to endothelial dysfunction in the metabolic syndrome. We observed reduced coronary arteriolar vasodilation to the endothelium-dependent dilator, acetylcholine (Ach), in Zucker Obese Fatty rats (ZOF, 34 ± 15% [mean ± standard deviation] 10-3 M) compared to Zucker Lean rats (ZLN, 98 ± 11%). This reduction in dilation occurred concomitantly with mitochondrial DNA (mtDNA) strand lesions and reduced mitochondrial complex activities in the endothelium of ZOF versus ZLN. To demonstrate endothelial dysfunction is linked to impaired mitochondrial function, administration of a cell-permeable, mitochondria-directed endonuclease (mt-tat-EndoIII), to repair oxidatively modified DNA in ZOF, restored mitochondrial function and vasodilation to Ach (94 ± 13%). Conversely, administration of a cell-permeable, mitochondria-directed exonuclease (mt-tat-ExoIII) produced mtDNA strand breaks in ZLN, reduced mitochondrial complex activities and vasodilation to Ach in ZLN (42 ± 16%). To demonstrate that mitochondrial function is central to endothelium-dependent vasodilation, we introduced (via electroporation) liver mitochondria (from ZLN) into the endothelium of a mesenteric vessel from ZOF and restored endothelium-dependent dilation to vasoactive intestinal peptide (VIP at 10-5 M, 4 ± 3% vasodilation before mitochondrial transfer and 48 ± 36% after transfer). Finally, to demonstrate mitochondrial function is key to endothelium-dependent dilation, we administered oligomycin (mitochondrial ATP synthase inhibitor) and observed a reduction in endothelium-dependent dilation. We conclude that mitochondrial function is critical for endothelium-dependent vasodilation.
Assuntos
Síndrome Metabólica , Vasodilatação , Acetilcolina/metabolismo , Acetilcolina/farmacologia , Animais , DNA Mitocondrial/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular , Síndrome Metabólica/metabolismo , Mitocôndrias/metabolismo , Ratos , Ratos ZuckerRESUMO
In vitro simulation of three-dimensional (3D) shoulder motion using in vivo kinematics obtained from human subjects allows investigation of clinical conditions in the context of physiologically relevant biomechanics. Herein, we present a framework for laboratory simulation of subject-specific kinematics that combines individual 3D scapular and humeral control in cadavers. The objectives were to: (1) robotically simulate seven healthy subject-specific 3D scapulothoracic and glenohumeral kinematic trajectories in six cadavers, (2) characterize system performance using kinematic orientation accuracy and repeatability, and muscle force repeatability metrics, and (3) analyze effects of input kinematics and cadaver specimen variability. Using an industrial robot to orient the scapula range of motion (ROM), errors with repeatability of ±0.1 mm and <0.5 deg were achieved. Using a custom robot and a trajectory prediction algorithm to orient the humerus relative to the scapula, orientation accuracy for glenohumeral elevation, plane of elevation, and axial rotation of <3 deg mean absolute error (MAE) was achieved. Kinematic accuracy was not affected by varying input kinematics or cadaver specimens. Muscle forces over five repeated setups showed variability typically <33% relative to the overall simulations. Varying cadaver specimens and subject-specific human motions showed effects on muscle forces, illustrating that the system was capable of differentiating changes in forces due to input conditions. The anterior and middle deltoid, specifically, showed notable variations in patterns across the ROM that were affected by subject-specific motion. This machine provides a platform for future laboratory studies to investigate shoulder biomechanics and consider the impacts of variable input kinematics from populations of interest, as they can significantly impact study outputs and resultant conclusions.
Assuntos
Articulação do Ombro , Ombro , Fenômenos Biomecânicos , Cadáver , Humanos , Úmero/fisiologia , Amplitude de Movimento Articular/fisiologia , Escápula/fisiologiaRESUMO
INTRODUCTION: Three-dimensional planning of humeral head osteotomy in shoulder arthroplasty (SA) is understudied. This study evaluated whether a standard osteotomy technique along the anterosuperior anatomic neck (ASOT) could be surgically reproduced as pre-operatively planned on 3D-CT models. MATERIAL AND METHODS: Pre-operative planning in 12 cadaver shoulders was performed on a 3D-CT model of the humerus to calculate the planned osteotomy plane (planned OP). The osteotomy was then performed using a free-hand technique, and a post-operative CT scan was obtained for analysis (performed OP). Planes were compared with regards to inclination, retroversion, and resected humeral head thickness so the accuracy could be quantified. RESULTS: The absolute errors between the performed and planned OP were 2° (0-10°), 5° (0-14°), and 4 mm (1-7 mm) for inclination, retroversion, and resected head thickness, respectively. Deviation < 10° for inclination and retroversion and < 5 mm for resected humeral head thickness between planned and performed OP was achieved in 92%, 83%, 58% of cases, respectively. No differences were found for inclination (p = 0.289), whereas retroversion and resected head thickness were smaller than planned (p ≤ 0.027). CONCLUSIONS: Pre-operative planning of the ASOT using a 3D-CT model is accurate within a threshold of 10° when using a free-hand technique in 92% of cases for inclination. Retroversion and resected head thickness differed from the pre-operative plan, thereby limiting the unrestricted use of humeral head osteotomy planning from 3D-CT models in SA. These findings are a reference for further studies to develop and quantify the accuracy of pre-operative planning software including cutting guides for SA using 3D-CT models. LEVEL OF EVIDENCE: Basic science article.
Assuntos
Artroplastia do Ombro , Articulação do Ombro , Artroplastia do Ombro/métodos , Humanos , Cabeça do Úmero/diagnóstico por imagem , Cabeça do Úmero/cirurgia , Osteotomia/métodos , Articulação do Ombro/diagnóstico por imagem , Articulação do Ombro/cirurgia , Tomografia Computadorizada por Raios XRESUMO
The connection between metabolism and flow in the heart, metabolic dilation, is essential for cardiac function. We recently found redox-sensitive Kv1.5 channels play a role in coronary metabolic dilation; however, more than one ion channel likely plays a role in this process as animals null for these channels still showed limited coronary metabolic dilation. Accordingly, we examined the role of another Kv1 family channel, the energetically linked Kv1.3 channel, in coronary metabolic dilation. We measured myocardial blood flow (contrast echocardiography) during norepinephrine-induced increases in cardiac work (heart rate x mean arterial pressure) in WT, WT mice given correolide (preferential Kv1.3 antagonist), and Kv1.3-null mice (Kv1.3-/- ). We also measured relaxation of isolated small arteries mounted in a myograph. During increased cardiac work, myocardial blood flow was attenuated in Kv1.3-/- and in correolide-treated mice. In isolated vessels from Kv1.3-/- mice, relaxation to H2 O2 was impaired (vs WT), but responses to adenosine and acetylcholine were equivalent to WT. Correolide reduced dilation to adenosine and acetylcholine in WT and Kv1.3-/- , but had no effect on H2 O2 -dependent dilation in vessels from Kv1.3-/- mice. We conclude that Kv1.3 channels participate in the connection between myocardial blood flow and cardiac metabolism.
Assuntos
Circulação Coronária , Canal de Potássio Kv1.3/fisiologia , Miocárdio/metabolismo , Animais , Circulação Coronária/efeitos dos fármacos , Camundongos , Bloqueadores dos Canais de Potássio/farmacologia , Fluxo Sanguíneo Regional/efeitos dos fármacos , Triterpenos/farmacologia , Vasodilatação/efeitos dos fármacosRESUMO
Ischemic heart disease is still the leading cause of death even with the advancement of pharmaceutical therapies and surgical procedures. Early vascularization in the ischemic heart is critical for a better outcome. Although stem cell therapy has great potential for cardiovascular regeneration, the ideal cell type and delivery method of cells have not been resolved. We tested a new approach of stem cell therapy by delivery of induced vascular progenitor cells (iVPCs) grown on polymer micro-bundle scaffolds in a rat model of myocardial infarction. iVPCs partially reprogrammed from vascular endothelial cells (ECs) had potent angiogenic potential and were able to simultaneously differentiate into vascular smooth muscle cells (SMCs) and ECs in 2D culture. Under hypoxic conditions, iVPCs also secreted angiogenic cytokines such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) as measured by enzyme-linked immunosorbent assay (ELISA). A longitudinal micro-scaffold made from poly(lactic-co-glycolic acid) was sufficient for the growth and delivery of iVPCs. Co-cultured ECs and SMCs aligned well on the micro-bundle scaffold similarly as in the vessels. 3D cell/polymer micro-bundles formed by iVPCs and micro-scaffolds were transplanted into the ischemic myocardium in a rat model of myocardial infarction (MI) with ligation of the left anterior descending artery. Our in vivo data showed that iVPCs on the micro-bundle scaffold had higher survival, and better retention and engraftment in the myocardium than free iVPCs. iVPCs on the micro-bundles promoted better cardiomyocyte survival than free iVPCs. Moreover, iVPCs and iVPC/polymer micro-bundles treatment improved cardiac function (ejection fraction and fractional shortening, endocardial systolic volume) measured by echocardiography, increased vessel density, and decreased infarction size [endocardial and epicardial infarct (scar) length] better than untreated controls at 8 weeks after MI. We conclude that iVPCs grown on a polymer micro-bundle scaffold are new promising approach for cell-based therapy designed for cardiovascular regeneration in ischemic heart disease.
Assuntos
Células Progenitoras Endoteliais/transplante , Ácido Láctico/química , Músculo Liso Vascular/transplante , Infarto do Miocárdio/cirurgia , Miocárdio/patologia , Miócitos de Músculo Liso/transplante , Neovascularização Fisiológica , Ácido Poliglicólico/química , Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Células Progenitoras Endoteliais/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Músculo Liso Vascular/metabolismo , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/metabolismo , Miócitos de Músculo Liso/metabolismo , Comunicação Parácrina , Fenótipo , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Ratos Sprague-Dawley , Transdução de Sinais , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Remodelação VentricularRESUMO
RATIONALE: In the working heart, coronary blood flow is linked to the production of metabolites, which modulate tone of smooth muscle in a redox-dependent manner. Voltage-gated potassium channels (Kv), which play a role in controlling membrane potential in vascular smooth muscle, have certain members that are redox-sensitive. OBJECTIVE: To determine the role of redox-sensitive Kv1.5 channels in coronary metabolic flow regulation. METHODS AND RESULTS: In mice (wild-type [WT], Kv1.5 null [Kv1.5(-/-)], and Kv1.5(-/-) and WT with inducible, smooth muscle-specific expression of Kv1.5 channels), we measured mean arterial pressure, myocardial blood flow, myocardial tissue oxygen tension, and ejection fraction before and after inducing cardiac stress with norepinephrine. Cardiac work was estimated as the product of mean arterial pressure and heart rate. Isolated arteries were studied to establish whether genetic alterations modified vascular reactivity. Despite higher levels of cardiac work in the Kv1.5(-/-) mice (versus WT mice at baseline and all doses of norepinephrine), myocardial blood flow was lower in Kv1.5(-/-) mice than in WT mice. At high levels of cardiac work, tissue oxygen tension dropped significantly along with ejection fraction. Expression of Kv1.5 channels in smooth muscle in the null background rescued this phenotype of impaired metabolic dilation. In isolated vessels from Kv1.5(-/-) mice, relaxation to H2O2 was impaired, but responses to adenosine and acetylcholine were normal compared with those from WT mice. CONCLUSIONS: Kv1.5 channels in vascular smooth muscle play a critical role in coupling myocardial blood flow to cardiac metabolism. Absence of these channels disassociates metabolism from flow, resulting in cardiac pump dysfunction and tissue hypoxia.
Assuntos
Circulação Coronária/fisiologia , Vasos Coronários/metabolismo , Canal de Potássio Kv1.5/fisiologia , Músculo Liso Vascular/metabolismo , Vasodilatação/fisiologia , Animais , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos TransgênicosRESUMO
BACKGROUND: The best chance that a shoulder arthroplasty will restore motion and muscle balance across the glenohumeral joint is by closely replicating natural articular morphology. Defining the humeral osteotomy plane along clear landmarks at the anatomic neck is critical. We hypothesized that a new osteotomy, based on alternative landmarks on the anatomic neck, would restore 3-dimensional humeral head morphology more reliably than the traditional osteotomy. METHODS: The anatomic neck was digitized in 30 human cadaver shoulders and compared with its 3-dimensional computed tomography reconstruction. Two different osteotomy techniques were virtually performed: the traditional, following the anterosuperior anatomic neck; and a new technique, defined by the inferoanterior anatomic neck. The length-width difference and orientation (retroversion, inclination) of the resection area were compared between the techniques and with native anatomy. RESULTS: Length-width difference of the anterosuperior resection area was higher than in the inferoanterior osteotomy (6 ± 2 mm vs. 3 ± 1 mm; P < .001). Retroversion of the anterosuperior resection plane was higher than the native head (50° ± 12° vs. 37° ± 11°; P < .001), whereas retroversion after the inferoanterior osteotomy (32° ± 12°) did not differ from native (P = .057). Inclination differed after the anterosuperior osteotomy (129° ± 5°) and the inferoanterior osteotomy (127° ± 4°) compared with the native head (134° ± 4°; P ≤ .001). CONCLUSION: The inferoanterior referenced osteotomy generated a more circular resection area, matching the native humeral head retroversion more closely than in the anterosuperior technique. This study suggests that in shoulder arthroplasty, the humeral resection level should be referenced at the inferoanterior rather than the anterosuperior anatomic neck. Further studies should investigate the biomechanical effects of this alternative resection plane.
Assuntos
Cabeça do Úmero/cirurgia , Artroplastia do Ombro/métodos , Cadáver , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteotomia/métodos , Tomografia Computadorizada por Raios XRESUMO
Mitochondrial dysfunction in obesity and diabetes can be caused by excessive production of free radicals, which can damage mitochondrial DNA. Because mitochondrial DNA plays a key role in the production of ATP necessary for cardiac work, we hypothesized that mitochondrial dysfunction, induced by mitochondrial DNA damage, uncouples coronary blood flow from cardiac work. Myocardial blood flow (contrast echocardiography) was measured in Zucker lean (ZLN) and obese fatty (ZOF) rats during increased cardiac metabolism (product of heart rate and arterial pressure, i.v. norepinephrine). In ZLN increased metabolism augmented coronary blood flow, but in ZOF metabolic hyperemia was attenuated. Mitochondrial respiration was impaired and ROS production was greater in ZOF than ZLN. These were associated with mitochondrial DNA (mtDNA) damage in ZOF. To determine if coronary metabolic dilation, the hyperemic response induced by heightened cardiac metabolism, is linked to mitochondrial function we introduced recombinant proteins (intravenously or intraperitoneally) in ZLN and ZOF to fragment or repair mtDNA, respectively. Repair of mtDNA damage restored mitochondrial function and metabolic dilation, and reduced ROS production in ZOF; whereas induction of mtDNA damage in ZLN reduced mitochondrial function, increased ROS production, and attenuated metabolic dilation. Adequate metabolic dilation was also associated with the extracellular release of ADP, ATP, and H2O2 by cardiac myocytes; whereas myocytes from rats with impaired dilation released only H2O2. In conclusion, our results suggest that mitochondrial function plays a seminal role in connecting myocardial blood flow to metabolism, and integrity of mtDNA is central to this process.
Assuntos
Vasos Coronários/fisiopatologia , DNA Mitocondrial/metabolismo , Síndrome Metabólica/fisiopatologia , Mitocôndrias/metabolismo , Animais , Vasos Coronários/metabolismo , Dano ao DNA/fisiologia , Fragmentação do DNA , Modelos Animais de Doenças , Síndrome Metabólica/metabolismo , Estresse Oxidativo/fisiologia , Ratos , Ratos Zucker , Espécies Reativas de Oxigênio/metabolismo , Vasodilatação/fisiologiaRESUMO
Ischemic heart disease (IHD) is a leading cause of death worldwide, and regenerative therapies through exogenous stem cell delivery hold promising potential. One limitation of such therapies is the vulnerability of stem cells to the oxidative environment associated with IHD. Accordingly, manipulation of stem cell mitochondrial metabolism may be an effective strategy to improve survival of stem cells under oxidative stress. MitoNEET is a redox-sensitive, mitochondrial target of thiazolidinediones (TZDs), and influences cellular oxidative capacity. Pharmacological targeting of mitoNEET with the novel TZD, mitoNEET Ligand-1 (NL-1), improved cardiac stem cell (CSC) survival compared to vehicle (0.1% DMSO) during in vitro oxidative stress (H2O2). 10 µM NL-1 also reduced CSC maximal oxygen consumption rate (OCR) compared to vehicle. Following treatment with dexamethasone, CSC maximal OCR increased compared to baseline, but NL-1 prevented this effect. Smooth muscle α-actin expression increased significantly in CSC following differentiation compared to baseline, irrespective of NL-1 treatment. When CSCs were treated with glucose oxidase for 7 days, NL-1 significantly improved cell survival compared to vehicle (trypan blue exclusion). NL-1 treatment of cells isolated from mitoNEET knockout mice did not increase CSC survival with H2O2 treatment. Following intramyocardial injection of CSCs into Zucker obese fatty rats, NL-1 significantly improved CSC survival after 24 h, but not after 10 days. These data suggest that pharmacological targeting of mitoNEET with TZDs may acutely protect stem cells following transplantation into an oxidative environment. Continued treatment or manipulation of mitochondrial metabolism may be necessary to produce long-term benefits related to stem cell therapies.
Assuntos
Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Células-Tronco/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Citometria de Fluxo , Masculino , Camundongos , Camundongos Knockout , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/citologia , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Zucker , Reação em Cadeia da Polimerase em Tempo Real , Células-Tronco/citologiaRESUMO
RATIONALE: A well-developed coronary collateral circulation improves the morbidity and mortality of patients following an acute coronary occlusion. Although regenerative medicine has great potential in stimulating vascular growth in the heart, to date there have been mixed results, and the ideal cell type for this therapy has not been resolved. OBJECTIVE: To generate induced vascular progenitor cells (iVPCs) from endothelial cells, which can differentiate into vascular smooth muscle cells (VSMCs) or endothelial cells (ECs), and test their capability to stimulate coronary collateral growth. METHODS AND RESULTS: We reprogrammed rat ECs with the transcription factors Oct4, Klf4, Sox2, and c-Myc. A population of reprogrammed cells was derived that expressed pluripotent markers Oct4, SSEA-1, Rex1, and AP and hemangioblast markers CD133, Flk1, and c-kit. These cells were designated iVPCs because they remained committed to vascular lineage and could differentiate into vascular ECs and VSMCs in vitro. The iVPCs demonstrated better in vitro angiogenic potential (tube network on 2-dimensional culture, tube formation in growth factor reduced Matrigel) than native ECs. The risk of teratoma formation in iVPCs is also reduced in comparison with fully reprogrammed induced pluripotent stem cells (iPSCs). When iVPCs were implanted into myocardium, they engrafted into blood vessels and increased coronary collateral flow (microspheres) and improved cardiac function (echocardiography) better than iPSCs, mesenchymal stem cells, native ECs, and sham treatments. CONCLUSIONS: We conclude that iVPCs, generated by partially reprogramming ECs, are an ideal cell type for cell-based therapy designed to stimulate coronary collateral growth.
Assuntos
Circulação Colateral , Circulação Coronária , Oclusão Coronária/cirurgia , Vasos Coronários/fisiopatologia , Células Endoteliais/transplante , Células-Tronco Pluripotentes Induzidas/transplante , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/transplante , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Oclusão Coronária/genética , Oclusão Coronária/metabolismo , Oclusão Coronária/patologia , Oclusão Coronária/fisiopatologia , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Epigênese Genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Camundongos , Camundongos SCID , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Neovascularização Fisiológica , Ratos , Ratos Sprague-Dawley , Medicina Regenerativa/métodos , Fluxo Sanguíneo Regional , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Teratoma/metabolismo , Teratoma/patologia , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transdução GenéticaRESUMO
OBJECTIVE: Our goal was to determine the mechanism by which mitochondrial oxidative stress impairs collateral growth in the heart. APPROACH AND RESULTS: Rats were treated with rotenone (mitochondrial complex I inhibitor that increases reactive oxygen species production) or sham-treated with vehicle and subjected to repetitive ischemia protocol for 10 days to induce coronary collateral growth. In control rats, repetitive ischemia increased flow to the collateral-dependent zone; however, rotenone treatment prevented this increase suggesting that mitochondrial oxidative stress compromises coronary collateral growth. In addition, rotenone also attenuated mitochondrial complex I activity and led to excessive mitochondrial aggregation. To further understand the mechanistic pathway(s) involved, human coronary artery endothelial cells were treated with 50 ng/mL vascular endothelial growth factor, 1 µmol/L rotenone, and rotenone/vascular endothelial growth factor for 48 hours. Vascular endothelial growth factor induced robust tube formation; however, rotenone completely inhibited this effect (P<0.05 rotenone versus vascular endothelial growth factor treatment). Inhibition of tube formation by rotenone was also associated with significant increase in mitochondrial superoxide generation. Immunoblot analyses of human coronary artery endothelial cells with rotenone treatment showed significant activation of adenosine monophosphate activated kinase (AMPK)-α and inhibition of mammalian target of rapamycin and p70 ribosomal S6 kinase. Activation of AMPK-α suggested impairments in energy production, which was reflected by decrease in O2 consumption and bioenergetic reserve capacity of cultured cells. Knockdown of AMPK-α (siRNA) also preserved tube formation during rotenone, suggesting the negative effects were mediated by the activation of AMPK-α. Conversely, expression of a constitutively active AMPK-α blocked tube formation. CONCLUSIONS: We conclude that activation of AMPK-α during mitochondrial oxidative stress inhibits mammalian target of rapamycin signaling, which impairs phenotypic switching necessary for the growth of blood vessels.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Vasos Coronários/enzimologia , Células Endoteliais/enzimologia , Mitocôndrias/metabolismo , Estresse Oxidativo/fisiologia , Transdução de Sinais/fisiologia , Animais , Peso Corporal/fisiologia , Células Cultivadas , Vasos Coronários/citologia , Modelos Animais de Doenças , Células Endoteliais/citologia , Humanos , Isquemia/metabolismo , Isquemia/patologia , Mitocôndrias/efeitos dos fármacos , Miocárdio/enzimologia , Miocárdio/patologia , Ratos , Ratos Endogâmicos WKY , Rotenona/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Desacopladores/farmacologiaRESUMO
Many clinical trials have attempted to use stem cells to treat ischemic heart diseases (IHD), but the benefits have been modest. Though coronary collaterals can be a "natural bypass" for IHD patients, the regulation of coronary collateral growth (CCG) and the role of endogenous stem cells in CCG are not fully understood. In this study, we used a bone marrow transplantation scheme to study the role of bone marrow stem cells (BMSCs) in a rat model of CCG. Transgenic GFP rats were used to trace BMSCs after transplantation; GFP bone marrow was harvested or sorted for bone marrow transplantation. After recovering from transplantation, the recipient rats underwent 10 days of repetitive ischemia (RI), with echocardiography before and after RI, to measure cardiac function and myocardial blood flow. At the end of RI, the rats were sacrificed for the collection of bone marrow for flow cytometry or heart tissue for imaging analysis. Our study shows that upon RI stimulation, BMSCs homed to the recipient rat hearts' collateral-dependent zone (CZ), proliferated, differentiated into endothelial cells, and engrafted in the vascular wall for collateral growth. These RI-induced collaterals improved coronary blood flow and cardiac function in the recipients' hearts during ischemia. Depletion of donor CD34+ BMSCs led to impaired CCG in the recipient rats, indicating that this cell population is essential to the process. Overall, these results show that BMSCs contribute to CCG and suggest that regulation of the function of BMSCs to promote CCG might be a potential therapeutic approach for IHD.
Assuntos
Circulação Colateral , Isquemia Miocárdica , Ratos , Animais , Circulação Colateral/fisiologia , Medula Óssea , Células Endoteliais , Isquemia Miocárdica/terapia , Isquemia , Células-TroncoRESUMO
Age affects gross shoulder range of motion (ROM), but biomechanical changes over a lifetime are typically only characterized for the humerothoracic joint. Suitable age-related baselines for the scapulothoracic and glenohumeral contributions to humerothoracic motion are needed to advance understanding of shoulder injuries and pathology. Notably, biomechanical comparisons between younger or older populations may obscure detected differences in underlying shoulder motion. Herein, biplane fluoroscopy and skin-marker motion analysis quantified humerothoracic, scapulothoracic, and glenohumeral motion during 3 static poses (resting neutral, internal rotation to L4-L5, and internal rotation to maximum reach) and 2 dynamic activities (scapular plane abduction and external rotation in adduction). Orientations during static poses and rotations during active ROM were compared between subjects <35 years and >45 years of age (N = 10 subjects per group). Numerous age-related kinematic differences were measured, ranging 5-22°, where variations in scapular orientation and motion were consistently observed. These disparities are on par with or exceed mean clinically important differences and standard error of measurement of clinical ROM, which indicates that high resolution techniques and appropriately matched controls are required to avoid confounding results of studies that investigate shoulder kinematics. Understanding these dissimilarities will help clinicians manage expectations and treatment protocols where indications and prevalence between age groups tend to differ. Where possible, it is advised to select age-matched control cohorts when studying the kinematics of shoulder injury, pathology, or surgical/physical therapy interventions to ensure clinically important differences are not overlooked.
Assuntos
Articulação do Ombro , Fenômenos Biomecânicos , Humanos , Amplitude de Movimento Articular , Escápula , OmbroRESUMO
OBJECTIVE: The purpose of this study was to determine whether G-CSF promotes coronary collateral growth (CCG) and decipher the mechanism for this stimulation. METHODS AND RESULTS: In a rat model of repetitive episodic myocardial ischemia (RI, 40 seconds LAD occlusion every 20 minutes for 2 hours and 20 minutes, 3 times/d for 5 days) CCG was deduced from collateral-dependent flow (flow to LAD region during occlusion). After RI, G-CSF (100 microg/kg/d) increased CCG (P<0.01) (0.47+/-0.15) versus vehicle (0.14+/-0.06). Surprisingly, G-CSF treatment without RI increased CCG (0.57+/-0.18) equal to G-CSF+RI. We evaluated ROS by dihydroethidine (DHE) fluorescence (LV injection, 60 microg/kg, during two episodes of ischemia). DHE fluorescence was double in G-CSF+RI versus vehicle+RI (P<0.01), and even higher in G-CSF without RI (P<0.01). Interestingly, the DHE signal did not colocalize with myeloperoxidase (immunostaining, neutrophil marker) but appeared in cardiac myocytes. The study of isolated cardiac myocytes revealed the cytokine stimulates ROS which elicit production of angiogenic factors. Apocynin inhibited G-CSF effects both in vivo and in vitro. CONCLUSIONS: G-CSF stimulates ROS production directly in cardiomyocytes, which plays a pivotal role in triggering adaptations of the heart to ischemia including growth of the coronary collaterals.
Assuntos
Circulação Colateral/fisiologia , Fator Estimulador de Colônias de Granulócitos/metabolismo , Isquemia Miocárdica/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Análise de Variância , Animais , Células Cultivadas , Circulação Colateral/efeitos dos fármacos , Circulação Coronária/efeitos dos fármacos , Modelos Animais de Doenças , Ecocardiografia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Testes de Função Cardíaca , Humanos , Imuno-Histoquímica , Masculino , Isquemia Miocárdica/diagnóstico por imagem , Isquemia Miocárdica/tratamento farmacológico , Isquemia Miocárdica/patologia , Miócitos Cardíacos/metabolismo , Probabilidade , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Valores de ReferênciaRESUMO
BACKGROUND: Interpretation of shoulder motion across studies has been complicated due to the use of numerous scapular coordinate systems in the literature. Currently, there are no simple means by which to compare scapular kinematics between coordinate system definitions when data from only one coordinate system is known. RESEARCH QUESTION: How do scapular kinematics vary based on the choice of coordinate system and can average rotation matrices be used to accurately convert kinematics between scapular local coordinate systems? METHODS: Average rotation matrices derived from anatomic landmarks of 51 cadaver scapulae (29â¯M/22â¯F; 59⯱â¯13 yrs; 26R/25â¯L; 171⯱â¯11â¯cm; 70⯱â¯19â¯kg; 23.7⯱â¯5.5â¯kg/m2) were generated between three common scapular coordinate systems. Absolute angle of rotation was used to determine if anatomical variability within the cadaver population influenced the matrices. To quantify the predictive capability to convert kinematics between the three coordinate systems, the average rotation matrices were applied to scapulothoracic motion data collected from 19 human subjects (10â¯M/9â¯F; 43⯱â¯17 yrs; 19R; 173⯱â¯9â¯cm; 71⯱â¯16â¯kg; 23.6⯱â¯4.5â¯kg/m2) using biplane fluoroscopy. Root mean squared error (RMSE) was used to compare kinematics from an original coordinate system to the kinematics expressed in each alternative coordinate system. RESULTS: The choice of scapular coordinate system resulted in mean differences in scapulothoracic rotation of up to 23°, with overall different shapes and/or magnitudes of the curves. A single average rotation matrix between any two coordinate systems achieved accurate conversion of scapulothoracic kinematics to within 4° of RMSE of the known solution. The average rotation matrices were independent of sex, side, decomposition sequence, and motion. SIGNIFICANCE: Scapulothoracic kinematic representations vary in shape and magnitude based solely on the choice of local coordinate system. The results of this study enhance interpretability and reproducibility in expressing scapulothoracic motion data between laboratories by providing a simple means to convert data between common coordinate systems. This is necessitated by the variety of available motion analysis techniques and their respective scapular landmark definitions.
Assuntos
Escápula/fisiopatologia , Fenômenos Biomecânicos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos TestesRESUMO
OBJECTIVE: We hypothesize that controversial effects of angiotensin II (Ang II) are attributable to its regulation of reactive oxygen species (ROS) and ROS-dependent signaling. METHODS AND RESULTS: Coronary collateral growth (CCG) was stimulated in normal (WKY) and syndrome X (JCR) rats by transient/repetitive ischemia (RI). Blood flow was measured in the normal (NZ) and the collateral-dependent (CZ) zone. In WKY, RI increased CZ flow (0.84 mL/min/g), but RI+subpressor Ang II increased it more (1.24 mL/min/g). This was associated with transient p38 and sustained Akt activation. A hypertensive dose of Ang II decreased CZ flow (0.69 mL/min/g), which was associated with sustained p38 and transient Akt activation. AT1R blockade by candesartan abrogated CZ flow in WKY (0.58 mL/min/g), reduced myocardial superoxide, and blocked p38 and Akt activation. RI-induced CZ flow in JCR was significantly decreased compared with WKY (0.12 mL/min/g), associated with a large increase in superoxide and lack of p38 and Akt activation. CZ flow in JCR was partially restored by candesartan (0.45 mL/min/g), accompanied by reduction in superoxide and partial restoration of p38 and Akt activation. CONCLUSIONS: Ang II/AT1R blockade, at least in part, regulates CCG via generating optimal ROS amounts and activating redox-sensitive signaling.
Assuntos
Angiotensina II/fisiologia , Oclusão Coronária/fisiopatologia , Isquemia/fisiopatologia , Neovascularização Fisiológica/fisiologia , Estresse Oxidativo/fisiologia , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Animais , Oclusão Coronária/tratamento farmacológico , Modelos Animais de Doenças , Hemorreologia , Isquemia/tratamento farmacológico , Masculino , Ratos , Espécies Reativas de Oxigênio/efeitos adversos , Espécies Reativas de Oxigênio/metabolismo , Receptor Tipo 1 de Angiotensina/fisiologia , Vasoconstritores/farmacologiaRESUMO
BACKGROUND: The goal of this study was to determine whether the expression of vascular endothelial growth factor (VEGF) is critical for coronary collateral growth. Previous studies have provided an association between coronary collateral growth and VEGF, but none have allowed determination of a causal role. METHODS AND RESULTS: We measured coronary collateral growth in rats subjected to repetitive episodes of myocardial ischemia (RI; one 40-second occlusion every 20 minutes for 2 hours 40 minutes, followed by 5 hours 20 minutes of rest, with this 8-hour cycle repeated 3 times per day for 10 days). Collateral growth was measured from blood flow (radioactive microspheres), visualization of arterial-arterial anastomoses (x-ray micro-CT), and maintenance of function during complete coronary occlusion in 3 groups of animals: sham (received instrumentation but no RI), experimental (subjected to RI), and anti-vascular endothelial growth factor (RI+anti-VEGF 0.6 mg/100 g per day) to block the endogenous actions of VEGF. In the 3 groups, native collateral flow (measurement for RI or sham protocol) averaged 0.2 to 0.3 mL x min(-1) x g(-1) of tissue. In the sham group, collateral flow did not increase during the protocol. Collateral flow in the control RI group increased by approximately 6-fold to 1.63 mL x min(-1) x g(-1) tissue, but in the anti-VEGF group, collateral flow did not increase after the RI protocol (0.22 mL x min(-1) x g(-1)). In acute experiments, collateral flow was unchanged during vasodilation with dipyridamole, indicating the increases in collateral flow are due to collateral growth and not vasodilation. X-ray micro-CT analysis revealed a 3-fold increase (versus sham group) in the number of arterial-arterial anastomoses per heart after RI, which was prevented by treatment with anti-VEGF. The growth of the collateral circulation was functional in the RI group because complete coronary occlusion did not induce any untoward effects on hemodynamics or arrhythmias. In the sham or anti-VEGF groups, coronary occlusion at the end of the protocol induced many arrhythmias and deterioration of function. CONCLUSIONS: From these results, we conclude that the expression of VEGF is critical to the growth of coronary collaterals.
Assuntos
Circulação Colateral/fisiologia , Vasos Coronários/crescimento & desenvolvimento , Fator A de Crescimento do Endotélio Vascular/sangue , Animais , Biomarcadores/sangue , Modelos Animais de Doenças , Masculino , Isquemia Miocárdica/sangue , Isquemia Miocárdica/fisiopatologia , Ratos , Ratos WistarRESUMO
Polyhydroxyalkanoate (PHA) mesh is a bioresorbable scaffold used to reinforce the suture-tendon interface in rotator cuff repairs (RCRs). We conducted a study of cyclic and ultimate failure properties of PHA mesh-augmented single-row RCRs and nonaugmented RCRs. Eight pairs of fresh-frozen cadaver humeri (6 male, 2 female) were tested. Mean (SD) age was 61 (9) years. The supraspinatus tendon was resected and reattached in a single-row configuration using 2 triple-loaded suture anchors and 6 simple stitches. The opposite humerus underwent RCR augmented with 2 strips of 13-mm × 23-mm PHA mesh. Humeri were mounted in an Instron load frame, cycled 1000 times to 1.0 MPa of effective stress, and loaded to failure. Construct gapping and ultimate failure loads/displacements were recorded. Paired t tests compared augmented and nonaugmented RCRs (P ≤ .05 was significant). There was no difference in gapping over 1000 cycles (P = .879). Mean (SD) failure load was higher for PHA mesh-augmented RCRs, 571 (173) N, than for nonaugmented (control) RCRs, 472 (120) N (P = .042), and failures were consistent within pairs because of tissue failure at the knots or anchor pullout. This technique for arthroscopic augmentation can be used to improve initial biomechanical repair strength in tears at risk for failure.