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Hyperbaric oxygen facilitates the effect of endothelial progenitor cell therapy on improving outcome of rat critical limb ischemia.

ABSTRACT: We tested the hypothesis that hyperbaric oxygen (HBO) (100% oxygen/2.4 atmospheres) facilitated the effect of autologous endothelial progenitor cell (EPC) therapy on restoring the blood flow in rat critical-limb ischemia (CLI). Adult-male-SD rats (n = 8/each group) were categorized into group 1 [sham control (SC)], group 2 (CLI-treated with culture medium), group 3 [CLI-intermittent HBO (3 h/day for 5 consecutive days after CLI), group 4 (CLI-EPC/2.0 × 10⁶ cells), and group 5 (CLI-HBO-EPC). By day 5 after CLI, flow cytometry showed that the circulating EPC (Sca-1/CD31+/C-kit/CD31+/CD34+) levels were highest in group 5 and lowest in group 2 (all P < 0.001). By day 14, laser Doppler demonstrated that the ratio of blood flow (i.e., CLI to normal hind-limb) was highest in group 1, lowest in group 2 and significantly higher in group 5 than in groups 3 and 4 (all P < 0.0001). The protein expressions of endothelial-cell biomarkers (CD31/vWF/eNOS), and numbers of endothelial-cell markers (CD31+/vWF+) and small vessels exhibited a similar pattern to blood-flow ratio among five groups, whereas the angiogenesis parameters in protein (CXCR4/SDF-1?/HIF-1?/VEGF) and cellular (HIF-1?/SDF-1?/CXCR4+) levels were progressively increased from groups 1 to 5 (all P < 0.0001). The protein expression of apoptotic (mitochondrial-Bax/cleaved-capspase-3/PARP), fibrotic (p-Smad3/TGF-ß) and mitochondrial-damaged (cytosolic-cytochrome C) exhibited an opposite pattern, whereas the protein expressions of anti-fibrotic (BMP-2/p-Smad1/5) and mitochondrial integrity (mitochondrial-cytochrome C) exhibited an identical pattern of ratio of blood flow among the five groups (all P < 0.0001). Combined HBO-EPC therapy is superior to either one alone in improving ischemia in rodent CLI.

SUBMITTER: Hsu SL

PROVIDER: S-EPMC6511789 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Hyperbaric oxygen facilitates the effect of endothelial progenitor cell therapy on improving outcome of rat critical limb ischemia.

Hsu Shan-Ling SL Yin Tsung-Cheng TC Shao Pei-Lin PL Chen Kuan-Hung KH Wu Re-Wen RW Chen Chien-Chang CC Lin Pao-Yuan PY Chung Sheng-Ying SY Sheu Jiunn-Jye JJ Sung Pei-Hsun PH Chen Chen-Yu CY Wang Ching-Jen CJ Yip Hon-Kan HK Lee Mel S MS

American journal of translational research 20190415 4

We tested the hypothesis that hyperbaric oxygen (HBO) (100% oxygen/2.4 atmospheres) facilitated the effect of autologous endothelial progenitor cell (EPC) therapy on restoring the blood flow in rat critical-limb ischemia (CLI). Adult-male-SD rats (n = 8/each group) were categorized into group 1 [sham control (SC)], group 2 (CLI-treated with culture medium), group 3 [CLI-intermittent HBO (3 h/day for 5 consecutive days after CLI), group 4 (CLI-EPC/2.0 × 10<sup>6</sup> cells), and group 5 (CLI-HBO ...[more]

PMID: 31105810

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Project description:BackgroundCritical limb ischemia (CLI) is characterized by lower extremity artery obstruction and a largely unexplained impaired ischemic neovascularization response. Bone marrow (BM) derived endothelial progenitor cells (EPC) contribute to neovascularization. We hypothesize that reduced levels and function of circulating progenitor cells and alterations in the BM contribute to impaired neovascularization in CLI.MethodsLevels of primitive (CD34(+) and CD133(+)) progenitors and CD34(+)KDR(+) EPC were analyzed using flow cytometry in blood and BM from 101 CLI patients in the JUVENTAS-trial (NCT00371371) and healthy controls. Blood levels of markers for endothelial injury (sE-selectin, sICAM-1, sVCAM-1, and thrombomodulin), and progenitor cell mobilizing and inflammatory factors were assessed by conventional and multiplex ELISA. BM levels and activity of the EPC mobilizing protease MMP-9 were assessed by ELISA and zymography. Circulating angiogenic cells (CAC) were cultured and their paracrine function was assessed.ResultsEndothelial injury markers were higher in CLI (P<0.01). CLI patients had higher levels of VEGF, SDF-1?, SCF, G-CSF (P<0.05) and of IL-6, IL-8 and IP-10 (P<0.05). Circulating EPC and BM CD34(+) cells (P<0.05), lymphocytic expression of CXCR4 and CD26 in BM (P<0.05), and BM levels and activity of MMP-9 (P<0.01) were lower in CLI. Multivariate regression analysis showed an inverse association between IL-6 and BM CD34(+) cell levels (P?=?0.007). CAC from CLI patients had reduced paracrine function (P<0.0001).ConclusionCLI patients have reduced levels of circulating EPC, despite profound endothelial injury and an EPC mobilizing response. Moreover, CLI patients have lower BM CD34(+)-cell levels, which were inversely associated with the inflammatory marker IL-6, and lower BM MMP-9 levels and activity. The results of this study suggest that inflammation-induced BM exhaustion and a disturbed progenitor cell mobilization response due to reduced levels and activity of MMP-9 in the BM and alterations in the SDF-1?/CXCR4 interaction contribute to the attenuated neovascularization in CLI patients.

Project description:INTRODUCTION:Open fractures with significant soft tissue injury are associated with high rates of complications, such as non-union, infection, chronic pain and disability. Complications often require further inpatient care, and in many cases, multiple operations and prolonged rehabilitation. Use of hyperbaric oxygen therapy as an adjunct to standard orthopaedic trauma care has the potential to reduce the complications of musculoskeletal injury and thus improve outcomes. Two previous randomised trials have suggested some positive effect, but neither functional measures nor long-term outcomes were reported. METHODS AND ANALYSIS:An international, multicentre, randomised, open-label, clinical trial. Patients with trauma with an acute open fracture of the tibia with severe soft tissue injury (Gustilo grade 3) and high risk of injury-related complications were recruited from participating major trauma hospitals with hyperbaric facilities. Patients were enrolled with the expectation of commencing 12 sessions of hyperbaric oxygen therapy within 48 h of injury. The primary outcome measure is the incidence of acute complications of the open fracture wound at 14 days. Other short-term outcome measures include amputation, need for fasciotomy, time until wound closure, breakdown of closed wounds, time until definitive orthopaedic fixation, number of operative procedures, intensive care stay and hospital stay. Long-term follow-up will continue for 2 years postinjury. ETHICS AND DISSEMINATION:Ethics approval was given by The Alfred Health Human Ethics Committee (206/04) and the Monash University Human Research Ethics Committee (CF07/4208). Approval was also obtained from the institutional research ethics committee at each participating site. This study will make a significant contribution to the trauma literature and should answer the question of whether hyperbaric oxygen therapy can significantly improve outcomes in severe lower limb trauma. Collective study results will be published in international journals and presented at relevant conferences. TRIAL REGISTRATION NUMBER:Clinicaltrials.gov: NCT00264511; Australian New Zealand Clinical Trials Registry (ANZCTR): ACTRN12607000559415.

Project description:Despite poor long-term patency, acceptable limb salvage has been reported with cryopreserved saphenous vein bypass (CVB) for various indications. However, utility of CVB in patients with critical limb ischemia (CLI) remains undefined. The purpose of this analysis was to determine the role of CVB in CLI patients and to identify predictors of successful outcomes.A retrospective review of all lower extremity bypass (LEB) procedures at a single institution was completed, and CVB in CLI patients were further analyzed. The primary end point was amputation-free survival. Secondary end points included primary patency and limb salvage. Life tables were used to estimate occurrence of end points. Cox regression analysis was used to determine predictors of limb salvage.From 2000 to 2012, 1059 patients underwent LEB for various indications, of whom 81 received CVB for either ischemic rest pain or tissue loss. Mean age (± standard deviation) was 66 ± 10 years (male, 51%; diabetes, 51%; hemodialysis dependence, 12%), and 73% (n = 59) had history of failed ipsilateral LEB or endovascular intervention. None had sufficient autogenous conduit for even composite vein bypass. Infrainguinal CVB (infrapopliteal target, 96%; n = 78) was completed for multiple indications including Rutherford class 4 (42%; n = 34), class 5 (40%; n = 32), and class 6 (18%; n = 15). Eleven (14%) had CLI and concomitant graft infection (n = 8) or acute on chronic ischemia (n = 3). Intraoperative adjuncts (eg, profundaplasty, suprainguinal stent or bypass) were completed in 49% (n = 40) of cases. Complications occurred in 36% (n = 29), with 30-day mortality of 4% (n = 3). Median follow-up for CLI patients was 11.8 (interquartile range, 0.4-28.4) months with corresponding 1- and 3-year actuarial estimated survival (± standard error mean) of 84% ± 4% and 62% ± 6%. Primary patency of CVB for CLI was 27% ± 6% and 17% ± 6% at 1 and 3 years, respectively. Amputation-free survival was 43% ± 6% and 23% ± 6% at 1 and 3 years, respectively, and significantly higher for rest pain (59% ± 9%, 36% ± 10%) compared with tissue loss (31% ± 7%, 14% ± 7%; log-rank, P = .04). Freedom from major amputation after CVB for CLI was 57% ± 6% and 43% ± 7% at 1 and 3 years. Multivariable predictors of limb salvage for the CVB CLI cohort included postoperative warfarin (hazard ratio [HR], 0.4; 95% confidence interval [CI], 0.2-0.8), dyslipidemia (HR, 0.4; 95% CI, 0.2-0.9), and rest pain (HR, 0.4; 95% CI, 0.2-0.9). Predictors of major amputation included graft infection (HR, 3.1; 95% CI, 1.1-9.0).In CLI patients with no autologous conduit and prior failed infrainguinal bypass, CVB outcomes are disappointing. CVB performs best in patients with rest pain, particularly those who can be anticoagulated with warfarin. However, it may be an acceptable option in patients with minor tissue loss or concurrent graft infection, but consideration should be weighed against the known natural history of nonrevascularized CLI and nonbiologic conduit alternatives, given potential cost implications.

Dataset Information

Hyperbaric oxygen facilitates the effect of endothelial progenitor cell therapy on improving outcome of rat critical limb ischemia.

Publications

Hyperbaric oxygen facilitates the effect of endothelial progenitor cell therapy on improving outcome of rat critical limb ischemia.

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