Adrecizumab, a non-neutralizing anti-adrenomedullin antibody, improves haemodynamics and attenuates myocardial oxidative stress in septic rats.

BACKGROUND: Sepsis still represents a major health issue, with persistent high morbidity and mortality rates. Cardiovascular dysfunction occurs frequently during sepsis. Adrenomedullin has been identified as a key mediator in vascular tone regulation. A non-neutralizing anti-adrenomedullin antibody, Adrecizumab, may improve haemodynamic dysfunction during caecal ligation and puncture-induced septic shock in a murine model. Our objective was to determine the role of Adrecizumab on haemodynamics in a rat model of sepsis. METHODS: For the induction of sepsis, caecal ligation and puncture were performed in Wistar male rats. Single blinded administration of Adrecizumab (2 mg/kg) or placebo was injected i.v. 24 h after the surgery, and norepinephrine was infused as the standard of care. There were > 7 animals per group. Invasive blood pressure and cardiac function (by echocardiography) were assessed until 3 h after Adrecizumab injection. RESULTS: A single therapeutic injection of Adrecizumab in septic rats induced rapid haemodynamic benefits with an increase in systolic blood pressure in septic-Adrecizumab rats versus untreated-septic rats (p = 0.049). The shortening fraction did not differ between the untreated-septic and septic-Adrecizumab groups. However, cardiac output increased during the 3 h after a single dose of Adrecizumab compared to untreated septic rats (p = 0.006). A single dose of Adrecizumab resulted in similar haemodynamics to the continuous administration of norepinephrine. Three hours after a single injection of Adrecizumab, there was no change in the inflammatory phenotype (TNFα, IL-10) in the hearts of the septic rats. By contrast, 3 h after a single Adrecizumab injection, free-radical production decreased in the hearts of septic-Adrecizumab vs untreated septic rats (p < 0.05). CONCLUSIONS: In a rat model of sepsis, a single therapeutic injection of Adrecizumab rapidly restored haemodynamic parameters and blunted myocardial oxidative stress. Currently, a proof-of-concept and dose-finding phase II trial (Adrenoss-2) is ongoing in patients with septic shock and elevated concentrations of circulating bio-adrenomedullin.

Effects of hypoestrogenism and/or hyperaldosteronism on myocardial remodeling in female mice.

We investigated the potential adverse effects of hyperaldosteronism and/or hypoestrogenism on cardiac phenotype, and examined their combined effects in female mice overexpressing cardiac aldosterone synthase (AS). We focused on some signaling cascades challenging defensive responses to adapt and/or to survive in the face of double deleterious stresses, such as Ca2+ -homeostasis, pro/anti-hypertrophic, endoplasmic reticulum stress (ER stress), pro- or anti-apoptotic effectors, and MAP kinase activation, and redox signaling. These protein expressions were assessed by immunoblotting at 9 weeks after surgery. Female wild type (FWT) and FAS mice were fed with phytoestrogen-free diet; underwent ovariectomy (Ovx) or sham-operation (Sham). Ovx increased gain weight and hypertrophy index. Transthoracic echocardiograghy was performed. Both Ovx-induced heart rate decrease and fractional shortening increase were associated with collagen type III shift. Cardiac estrogen receptor (ERα, ERß) protein expression levels were downregulated in Ovx mice. Hypoestrogenism increased plasma aldosterone and MR protein expression in FAS mice. Both aldosterone and Ovx played as mirror effects on up and downstream signaling effectors of calcium/redox homeostasis, apoptosis, such as concomitant CaMKII activation and calcineurin down-regulation, MAP kinase inhibition (ERK1/2, p38 MAPK) and Akt activation. The ratio Bcl2/Bax is in favor to promote cell survivor. Finally, myocardium had dynamically orchestrated multiple signaling cascades to restore tolerance to hostile environment thereby contributing to a better maintenance of Ca2+ /redox homeostasis. Ovx-induced collagen type III isoform shift and its upregulation may be important for the biomechanical transduction of the heart and the recovery of cardiac function in FAS mice. OVX antagonized aldosterone signaling pathways.

Akt-mediated cardioprotective effects of aldosterone in type 2 diabetic mice.

Studies have shown that aldosterone would have angiogenic effects and therefore would be beneficial in the context of cardiovascular diseases. We thus investigated the potential involvement of aldosterone in triggering a cardiac angiogenic response in the context of type-2 diabetes and the molecular pathways involved. Male 3-wk-old aldosterone synthase (AS)-overexpressing mice and their control wild-type (WT) littermates were fed a standard or high-fat, high-sucrose (HFHS) diet. After 6 mo of diet treatment, mice were euthanized, and cardiac samples were assayed by RT-PCR, immunoblotting, and immunohistology. HFHS diet induced type-2 diabetes in WT (WT-D) and AS (AS-D) mice. VEGFa mRNAs decreased in WT-D (-43%, P<0.05 vs. WT) and increased in AS-D mice (+236%, P< 0.01 vs. WT-D). In WT-D mouse hearts, the proapoptotic p38MAPK was activated (P<0.05 vs. WT and AS-D), whereas Akt activity decreased (-64%, P<0.05 vs. WT). The AS mice, which exhibited a cardiac up-regulation of IGF1-R, showed an increase in Akt phosphorylation when diabetes was induced (P<0.05 vs. WT and AS-D). Contrary to WT-D mice, AS-D mouse hearts did not express inflammatory markers and exhibited a normal capillary density (P<0.05 vs. WT-D). To our knowledge, this is the first study providing new insights into the mechanisms whereby aldosterone prevents diabetes-induced cardiac disorders.

Crucial role for endoplasmic reticulum stress during megakaryocyte maturation.

OBJECTIVE: Apoptotic-like phase is an essential step for the platelet formation from megakaryocytes. How controlled is this signaling pathway remained poorly understood. The aim of this study was to determine whether endoplasmic reticulum (ER) stress-induced apoptosis occurs during thrombopoiesis. APPROACH AND RESULTS: Investigation of ER stress and maturation markers in different models of human thrombopoiesis (CHRF, DAMI, MEG-01 cell lines, and hematopoietic stem cells: CD34(+)) as well as in immature pathological platelets clearly indicated that ER stress occurs transiently during thrombopoiesis. Direct ER stress induction by tunicamycin, an inhibitor of N-glycosylation, or by sarco/endoplasmic reticulum Ca(2+) ATPase type 3b overexpression, which interferes with reticular calcium, leads to some degree of maturation in megakaryocytic cell lines. On the contrary, exposure to salubrinal, a phosphatase inhibitor that prevents eukaryotic translation initiation factor 2α-P dephosphorylation and inhibits ER stress-induced apoptosis, decreased both expression of maturation markers in MEG-01 and CD34(+) cells as well as numbers of mature megakaryocytes and proplatelet formation in cultured CD34(+) cells. CONCLUSIONS: Taken as a whole, our research suggests that transient ER stress activation triggers the apoptotic-like phase of the thrombopoiesis process.

Role of L-type calcium channel blocking in epidermal growth factor receptor-independent activation of extracellular signal regulated kinase 1/2.

OBJECTIVE: Vascular smooth muscle cell (VSMC) differentiation, growth and survival, key events in the development of cardiovascular diseases, are under the control of signaling enzymes including extracellular signal regulated kinase 1/2 (ERK 1/2), Akt and epidermal growth factor receptor (EGFR) activation. EGFR trans-activation is known to mediate thrombin- or angiotensin II (AII)-stimulated ERK 1/2 activation. However, our laboratory has demonstrated, in thrombin-stimulated VSMC, that the prevention of intracellular Ca2+ elevation ([Ca2+]i) by BAPTA-AM pretreatment unveiled EGFR-independent ERK 1/2 activation. Since calcium channel blockers (CCBs) also impair agonist-induced [Ca2+]i elevation, we investigated whether EGFR-independent ERK 1/2 activation could occur in VSMCs treated by CCBs such as amlodipine, isradipine and verapamil, and examined the possible role of Akt. METHODS: Cultured VSMCs were pretreated or not with CCBs and with various inhibitors of the signaling pathways under study, prior to stimulation by thrombin or AII, and the phosphorylation/activation status of EGFR, Akt and ERK 1/2 was determined by Western blotting using phospho-specific antibodies. RESULTS AND CONCLUSION: Unlike BAPTA, CCBs did not impair stimulus-induced EGFR trans-activation, hence ERK1/2 phosphorylation. However, when EGFR kinase was inhibited, CCBs and BAPTA dose-dependently protected stimulus-induced ERK1/2 phosphorylation. The effect of amlodipine could not be mimicked by its R+ enantiomer, which is devoid of CCB activity, suggesting that the effects of CCBs were accounted for by their L-type Ca2+ channel-blocking property. Altogether, our results indicated that in G-protein-coupled receptor (GPCR)-stimulated VSMCs, the prevention of [Ca2+]i elevation by CCBs unmasked an EGFR kinase-independent phosphorylation of ERK 1/2. Since EGFR kinase inhibitors are supposed to be efficient in the treatment of some cancers, such a mechanism might be clinically relevant in hypertensive patients with cancer.

Evidence for ERK1/2 activation by thrombin that is independent of EGFR transactivation.

Thrombin is involved in abnormal proliferation of vascular smooth muscle cells (VSMCs) associated with pathogenic vascular remodeling. Thrombin stimulation results in extracellular signal-regulated kinase (ERK)1/2 activation through transactivation of the epidermal growth factor receptor (EGFR). Here, using specific antibodies and inhibitors, we investigated the thrombin-induced phosphorylation of Src family kinases, nonreceptor proline-rich tyrosine kinase (Pyk2), EGFR, and ERK1/2. Our results show that Src and Pyk2 are involved upstream of the EGFR transactivation that is required for ERK1/2 phosphorylation. The investigation of the role of intracellular calcium concentration ([Ca2+]i) and calcium mobilization with the Ca2+ chelator BAPTA and thapsigargin, respectively, indicated that thrombin- and thapsigargin-induced phosphorylation of the EGFR but not ERK1/2 is dependent on an increase in [Ca2+]i. Moreover, only after BAPTA-AM pretreatment was thrombin-induced activation of ERK1/2 partially preserved from the effects of EGFR and PKC inhibition but not Src family kinase inhibition. These results suggest that BAPTA, by preventing [Ca2+]i elevation, unmasks a new pathway of Src family kinase-dependent thrombin-stimulated ERK1/2 phosphorylation that is independent of EGFR and PKC activation.