AngiotensinII Preconditioning Promotes Angiogenesis In Vitro via ERKs Phosphorylation

AngiotensinII (AngII) is involved in not only the formation of cardiac hypertrophy but also the development of cardiac remodeling both of which are associated with myocardial angiogenesis. This study was therefore performed to clarify the effects of AngII on the formation of vasculatures by cultured cardiac microvascular endothelial cells (CMVECs) after a long-period stimulation with or without the AngII preconditioning. Incubation with AngII for 18 hrs significantly impaired the formation of capillary-like tubes comparing to that without AngII. CMVECs with AngII pretreatment for 5 and 10 min formed more capillary-like tubes than those without AngII pretreatment, suggesting that preconditioning with AngII at a lower dose for a short period could prevent the further damage of CMVECs by a higher concentration of AngII. Moreover, AngII (10−7 M) stimulation for 5 and 10 min significantly induced the increase in extracellular signal-regulated protein kinases (ERKs) phosphorylation, and an ERKs inhibitor, PD98059, abrogated the increase in the formation of capillary-like tubes induced by the AngII-pretreatment. In conclusion, preconditioning with a lower concentration of AngII for a short period prevents the subsequent impairment of CMVECs by a higher dose of AngII, at least in part, through the increase in ERKs phosphorylation

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

VEGFR endocytosis regulates the angiogenesis in a mouse model of hindlimb ischemia

Background: The regulation of angiogenesis in the treatment of cardiovascular diseases has been widely studied and the vascular endothelial growth factor (VEGF) families and VEGF receptor (VEGFR) have been proven to be one of the key regulators. The VEGFR endocytosis has been recently proved to be involved in the regulation of angiogenesis. Our previous study showed that the upregulation of VEGFR endocytosis enhanced angiogenesis in vitro. In this research, we utilized mice with induced hindlimb ischemia, as a model to investigate the role of VEGFR endocytosis in the regulation of angiogenesis in vivo. Our goal was to observe the effect of revascularization with different degrees of VEGFR endocytosis after injecting atypical protein kinase C inhibitor (uPKCi) and dynasore, which could respectively promote and inhibit the VEGFR endocytosis.Methods: We induced the hindlimb ischemia in adult male mice by ligating the hindlimb artery. By directly injecting the ischemic muscles with endothelial progenitor cells (EPCs) alone or EPCs + alpha PKCi/EPCs + dynasore or control medium (sham group), we divided the mice into four groups and detected lower limb blood flow using a laser Doppler blood perfusion imager. We also measured the immunohistochemistry (IHC) of markers for angiogenesis, such as CD31 and alpha smooth muscle actin (alpha-SMA) in the ischemic hindlimb tissues.Results: We demonstrated VEGFR endocytosis played an important role in the angiogenesis of the ischemic hindlimb model in vivo. By using atypical PKC inhibitor that increase the VEGFR endocytosis, the angiogenesis in the mice model was promoted. Treatment with EPCs + alpha PKCi showed greater effects on blood perfusion recovery and increased the alpha-SIVIA-positive vessels.Conclusions: The regulation of VEGFR endocytosis represents a valuable method of improving angiogenesis and thus revascularization in ischemic disease model

Mitochondrial aldehyde dehydrogenase-2 deficiency compromises therapeutic effect of ALDH bright cell on peripheral ischemia

The autologous ALDH bright (ALDHbr) cell therapy for ischemic injury is clinically safe and effective, while the underlying mechanism remains elusive. Here, we demonstrated that the glycolysis dominant metabolism of ALDHbr cells is permissive to restore blood flow in an ischemic hind limb model compared with bone marrow mononuclear cells (BMNCs). PCR array analysis showed overtly elevated Aldh2 expression of ALDHbr cells following hypoxic challenge. Notably, ALDHbr cells therapy induced blood flow recovery in this model was reduced in case of ALDH2 deficiency. Moreover, significantly reduced glycolysis flux and increased reactive oxygen species (ROS) levels were detected in ALDHbr cell from Aldh2-/- mice. Compromised effect on blood flow recovery was also noticed post transplanting the human ALDHbr cell from ALDH2 deficient patients (GA or AA genotypes) in this ischemic hindlimb mice model. Taken together, our findings illustrate the indispensable role of ALDH2 in maintaining glycolysis dominant metabolism of ALDHbr cell and advocate that patient's Aldh2 genotype is a prerequisite for the efficacy of ALDHbr cell therapy for peripheral ischemia

Regulation of p53 by Jagged1 Contributes to Angiotensin II-Induced Impairment of Myocardial Angiogenesis

<div><p>Angiotensin II (AngII) is a major contributor to the development of heart failure, however, the molecular and cellular mechanisms still remain elucidative. Inadequate angiogenesis in myocardium leads to transition from cardiac hypertrophy to dysfunction, this study was therefore conducted to examine the effects of AngII on myocardial angiogenesis and the underlying mechanisms. AngII treatment significantly impaired angiogenetic responses, which were determined by counting the capillaries either in matrigel formed by cultured cardiac microvascular endothelial cells (CMVECs) or in myocardium of mice and by measuring the <i>in vitro</i> and <i>in vivo</i> production of VEGF proteins, and stimulated accumulation and phosphorylation of cytosolic p53 which led to increases in phosphorylated p53 and decreases of hypoxia inducible factor (Hif-1) in nucleus. All of these cellular and molecular events induced by AngII in CEMCs and hearts of mice were largely reduced by a p53 inhibitor, pifithrin-α (PFT-α). Interestingly, AngII stimulated the upregulation of Jagged1, a ligand of Notch, but it didn’t affect the expression of Delta-like 4 (Dll-4), another ligand of Notch. Inhibition of p53 by PFT-α partly abolished this effect of AngII. Further experiments showed that knockdown ofJagged1 by addition of siRNA to cultured CMVECs dramatically declined AngII-stimulated accumulation and phosphorylation of p53 in cytosol, upregulation of phosphorylated p53 and downregulation of Hif-1 expression in nucleus, decrease of VEGF production and impairment of capillary-like tube formation by the cells. Our data collectively suggest that AngII impairs myocardial angiogenetic responses through p53-dependent downregulation of Hif-1 which is regulated by Jagged1/Notch1 signaling<i>.</i></p> </div

AngII-induced impairment of angiogenetic responses and accumulation and phosphorylation of p53 in cultured CMVECs.

<p>(<b>A</b>) Formation of capillary-like tubes. CMVECs were seeded onto the matrigel in plates containing 1 µM AngII (AngII) or vehicle (Control). Eighteen hours later, the formation of capillary-like tubes was observed under an optical microscope. <i>Left </i><i>panel</i>: Representative photomicrographs of capillary-like tubes (scale bar: 100 µm). <i>Right </i><i>panel</i>: Quantitative analysis for capillary-like tube formation. Capillary-like tubes were counted in randomly selected 5 fields for each plate. Data are expressed as mean ± S.E.M. obtained from 15 independent experiments (n=15). (<b>B</b>) Measurement of VEGF protein levels in culture medium. Cultured CMVECs were incubated with AngII (1 µM) or vehicle (Control) for 18 hours. Culture medium was collected and VEGF proteins were measured by ELISA. Data are expressed as mean ± S.E.M. obtained from 5 independent experiments (n=5). (<b>C</b>) Cytosolic p53 and p-p53 protein expression. CMVECs were treated as described under (B) and then collected and lysed for Western blot analyses of p53 and p-p53. β-actin was used as a loading control. (<b>D</b>) Nucleus p-p53 and Hif-1α expression. Nucleus proteins isolated from CMVECs treated as described under (B) were subjected to Western blot analyses for p-p53 and Hif-1α. TFIIB was used as a loading control. Representative immunoblots are shown. The expression of p53, p-p53 or Hif-1α was quantified as folds of β-actin, p53 or TFIIB, respectively. Data represent mean ± S.E.M. obtained from 3 independent experiments (n=3). ^*<i>p</i> < 0.05 <i>vs</i> Control.</p

Effect of Jagged1 siRNA on AngII-induced accumulation and phosphorylation of p53 and dowregulation of Hif-1α in CMVECs.

<p>(<b>A</b>) Downregulation of Jagged1 by siRNA in CMVECs. Three sequences of siRNA of Jagged1 (siRNA-Jagged1, 1 through 3) or scramble RNA were transfected to cultured CMVECs and then the cells were incubated with (AngII) or without (Cont) AngII (1 µM) for 18 hours. Jagged1 expression was detected by Western blotting. β-Actin expression served as a loading control. Representative immunoblots from 3 independent experiments are shown. (<b>B</b>) Inhibition of cytosolic p53 and p-p53 expression by siRNA of Jagged1. CMVECs transfected by #2 sequence of Jagged1 siRNA (si-Jag1) or scramble RNA (Scram) were stimulated with or without AngII (1µM) for 18 hours. Total cell lysate was subjected to Western blotting. β-Actin was used as a loading control. (<b>C</b>) Effect of Jagged1 siRNA on nucleus p-p53 and Hif-1α expression. Nucleus proteins isolated from CMVECs treated as described under (B) were subjected to Western blot analyses. TFIIB expression served as a loading control. Representative immunoblots are shown. The expression of p53, p-p53 or Hif-1α was quantified as folds of β-actin or of TFIIB, respectively. (<b>D</b>) Effect of Jagged1 siRNA on Hif-1 activity. (<b>E</b>) Effect of Jagged1 siRNA on notch/RBPj activity. HUVECs were transfected with Hif-1-luc or RBPj-Luc construct together with β-gal expression plasmid following by si-Jag1 transfection. 24 hour later, the cells were treated with Ang II for 24 hour and subjected for Luc assays. Data represent mean ± S.E.M. obtained from 3 independent experiments (n=3). ^*<i>p</i> < 0.05, ^**<i>p</i> < 0.01 <i>vs</i> Scram; ^#<i>p</i> < 0.05, ^{# #}<i>p</i> < 0.01 <i>vs</i> AngII+Scram.</p