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Bmal1 Downregulation Worsens Critical Limb Ischemia by Promoting Inflammation and Impairing Angiogenesis.

ABSTRACT: Critical limb ischemia (CLI) is the most advanced clinical stage of peripheral vascular disease with high mobility and mortality. CLI patients suffer from lower extremity rest pain, ulceration, and gangrene caused by insufficient blood and oxygen supply. Seeking for effective biomarkers and therapeutic targets is of great significance for improving the life quality of CLI patients. The circadian clock has been reported to be involved in the progression of kinds of cardiovascular diseases. Whether and how circadian genes play a role in CLI remains unknown. In this study, by collecting femoral artery and muscle specimens of CLI patients who underwent amputation, we confirmed that the circadian gene Bmal1 is downregulated in the CLI femoral artery and ischemic distal lower limb muscle. Furthermore, we verified that Bmal1 affects CLI by regulating lipid metabolism, inflammation, and angiogenesis. A hindlimb ischemia model performed in wild-type and Bmal1^-/- mice confirmed that Bmal1 disruption would lead to impaired angiogenesis. In vitro experiments indicated that the decreased expression of Bmal1 would increase ox-LDL uptake and impair endothelial cell functions, including proliferation, migration, and tube formation. As for mechanisms, Bmal1 represses inflammation by inhibiting lipid uptake and by activating IL-10 transcription and promotes angiogenesis by transcriptionally regulating VEGF expression. In conclusion, we provide evidence that the circadian gene Bmal1 plays an important role in CLI by inhibiting inflammation and promoting angiogenesis. Thus, Bmal1 may be an effective biomarker and a potential therapeutic target in CLI.

SUBMITTER: Xu L

PROVIDER: S-EPMC8384109 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundPrevious work demonstrated that high-fat diet (HFD) triggered thioredoxin-interacting protein (TXNIP) and that silencing TXNIP prevents diabetes-impaired vascular recovery. Here, we examine the impact of genetic deletion of TXNIP on HFD-impaired vascular recovery using hind limb ischemia model.MethodsWild type mice (WT, C57Bl/6) and TXNIP knockout mice (TKO) were fed either normal chow diet (WT-ND and TKO-ND) or 60% high-fat diet (WT-HFD and TKO-HFD). After four weeks of HFD, unilateral hind limb ischemia was performed and blood flow was measured using Laser doppler scanner at baseline and then weekly for an additional three weeks. Vascular density, nitrative stress, infiltration of CD68+ macrophages, and expression of inflammasome, vascular endothelial growth factor (VEGF), VEGF receptor-2 were examined by slot blot, Western blot and immunohistochemistry.ResultsBy week 8, HFD caused similar increases in weight, cholesterol and triglycerides in both WT and TKO. At week 4 and week 8, HFD significantly impaired glucose tolerance in WT and to a lesser extent in TKO. HFD significantly impaired blood flow and vascular density (CD31 labeled) in skeletal muscle of WT mice compared to ND but not in TKO. HFD and ischemia significantly induced tyrosine nitration, and systemic IL-1? and infiltration of CD68+ cells in skeletal muscle from WT but not from TKO. HFD significantly increased cleaved-caspase-1 and IL-1 ? compared to ND. Under both ND, ischemia tended to increase VEGF expression and increased VEGFR2 activation in WT only but not TKO.ConclusionSimilar to prior observation in diabetes, HFD-induced obesity can compromise vascular recovery in response to ischemic insult. The mechanism involves increased TXNIP-NLRP3 (nucleotide-binding oligomerization domain-like receptor protein 3) inflammasome activation, nitrative stress and impaired VEGFR2 activation. Deletion of TXNIP restored blood flow, reduced nitrative stress and blunted inflammasome-mediated inflammation; however, it did not impact VEGF/VEGFR2 in HFD. Targeting TXNIP-NLRP3 inflammasome can provide potential therapeutic target in obesity-induced vascular complication.

Project description:BackgroundAlthough the mononuclear cell (MNC) transplantation could theoretically induce therapeutic angiogenesis in the patients with no-option critical limb ischemia (NO-CLI), the clinical responses to this approach are inconsistent among different clinical trials. The purpose of this study was to identify the prognostic factors of responders and develop a predictive nomogram to guide patient selection.MethodsWe retrospectively reviewed a consecutive NO-CLI cohort who received peripheral blood-derived transplantation in our center. The patients who survived and achieved complete remission of CLI at 6 months post-transplantation were defined as responders. Logistic regression models were used to screen and identify the prognostic factors based on which predictive nomogram was developed. A receiver operating characteristic (ROC) curve and a calibration curve were drawn to determine the discrimination level and predictive accuracy.ResultsThe study ultimately enrolled 103 patients, including 58 responders and 45 non-responders. Based on the multivariate regression analysis, age ≥ 50 years (odds ratio [OR] 0.201, P = 0.004), blood fibrinogen > 4 g/L (OR 0.176, P = 0.003), arterial occlusion above the knee/elbow (OR 0.232, P = 0.010), the transcutaneous pressure of oxygen (TcPO2) (OR 1.062, P = 0.006), and the Log total transplanted CD34+ cell count (OR 3.506, P = 0.046) were identified as independent prognostic factors of the responders in the nomogram. An area under the ROC curve of 0.851 indicated good discrimination, and the calibration curve of the predicted probability showed optimal agreement with that of the observed probability.ConclusionsAge, blood fibrinogen, arterial occlusion level, TcPO2, and the total transplanted CD34+ cell count were independent prognostic factors of the responders. A nomogram with high discrimination and accuracy was developed to provide individualized predictions.Trail registrationChiCTR, ChiCTR1800019401 . Registered 9 November 2018-Retrospectively registered.

Project description:(1) This study tests hypothesis whether extracorporeal shock wave (ECSW) therapy effectively salvages mouse critical limb ischemia (CLI). In vitro result demonstrated that the angiogenesis parameters (i.e., tubular length/cluster/network formation) and protein expressions of EGFR/VEGFR2/RAS/c-Raf/MEK/ERK/VEGF/p-PI3K/p-Akt/p-m-TOR were significantly and progressively increased with stepwise augmentation of ECSW energy (0.1/0.14/0.20 mJ/mm2/140 impulses). On the other hand, they were suppressed by administration of Avastin (20 μM). Adult male B6 mice (n = 24) were equally categorized into group 1 (sham-operated control), group 2 (CLI), group 3 [CLI + ECSW (0.12 mJ/mm2/120 impulses/at days 1/3/7 after CLI induction)] and group 4 [CLI + ECSW (0.12 mJ/mm2/120 impulses) + Avastin (1 mg/intramuscular-injection)] at days 1/3/7 after CLI induction] and quadriceps were harvested by day 14. The laser Doppler result showed that the ratio of left (ischemia) to right (normal) limb blood flow was highest in group 1, lowest in group 2, and significantly higher in group 3 than in group 4 by days 7/14 after the CLI procedure (p < 0.0001). The protein expressions of cell proliferation/migration/angiogenesis receptors (EGFR/VEGFR2), angiogenesis biomarkers (VEGF/CXCR4/SDF-1) and cell proliferation/growth/survival (Ras/c-Raf/MEK/ERK)/(PI3K/Akt/m-TOR) and cell motility/proliferation (p-FAK/p-Scr) signaling biomarkers were significantly higher in group 3 than in groups 1/2/4, and significantly lower in group 1 than in groups 2/4, but they did not show a difference between groups 2 and 4 (all p < 0.001). The small vessel density and cellular levels of endothelial cell surface marker (CD31+) exhibited an identical pattern of blood flow, whereas the angiogenesis (CXCR4+/VEGF+) displayed an identical pattern of VEGFR2 among the groups (all p < 0.0001). The in vitro and in vivo studies found ECSW salvaged the CLI mainly through upregulating Ras-Raf-MEK/ERK/cell motility, cell proliferation/growth pathways and angiogenesis.

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Bmal1 Downregulation Worsens Critical Limb Ischemia by Promoting Inflammation and Impairing Angiogenesis.

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