MK5 haplodeficiency decreases collagen deposition and scar size during post-myocardial infarction wound repair.

MK5 is a protein serine/threonine kinase activated by p38, ERK3, and ERK4 MAPKs. MK5 mRNA and immunoreactivity are detected in mouse cardiac fibroblasts, and MK5 haplodeficiency attenuates the increase in collagen 1-α1 mRNA evoked by pressure overload. The present study examined the effect of MK5 haplodeficiency on reparative fibrosis following myocardial infarction (MI). Twelve-week-old MK5+/- and wild-type littermate (MK5+/+) mice underwent ligation of the left anterior descending coronary artery (LADL). Surviving mice were euthanized 8 or 21 days post-MI. Survival rates did not differ significantly between MK5+/+ and MK5+/- mice, with rupture of the LV wall being the primary cause of death. Echocardiographic imaging revealed similar increases in LV end-diastolic diameter, myocardial performance index, and wall motion score index in LADL-MK5+/+ and LADL-MK5+/- mice. Area at risk did not differ between LADL-MK5+/+ and LADL-MK5+/- hearts. In contrast, infarct size, scar area, and scar collagen content were reduced in LADL-MK5+/- hearts. Immunohistochemical analysis of mice experiencing heart rupture revealed increased MMP-9 immunoreactivity in the infarct border zone of LADL-MK5+/- hearts compared with LADL-MK5+/+. Although inflammatory cell infiltration was similar in LADL-MK5+/+ and LADL-MK5+/- hearts, angiogenesis was more pronounced in the infarct border zone of LADL-MK5+/- mice. Characterization of ventricular fibroblasts revealed reduced motility and proliferation in fibroblasts isolated from MK5-/- mice compared with those from both wild-type and haplodeficient mice. siRNA-mediated knockdown of MK5 in fibroblasts from wild-type mice also impaired motility. Hence, reduced MK5 expression alters fibroblast function and scar morphology but not mortality post-MI. NEW & NOTEWORTHY MK5/PRAK is a protein serine/threonine kinase activated by p38 MAPK and/or atypical MAPKs ERK3/4. MK5 haplodeficiency reduced infarct size, scar area, and scar collagen content post-myocardial infarction. Motility and proliferation were reduced in cultured MK5-null cardiac myofibroblasts.

VEGF and angiopoietins promote inflammatory cell recruitment and mature blood vessel formation in murine sponge/Matrigel model.

A key feature in the induction of pathological angiogenesis is that inflammation precedes and accompanies the formation of neovessels as evidenced by increased vascular permeability and the recruitment of inflammatory cells. Previously, we and other groups have shown that selected growth factors, namely vascular endothelial growth factor (VEGF) and angiopoietins (Ang1 and Ang2) do not only promote angiogenesis, but can also induce inflammatory response. Herein, given a pro-inflammatory environment, we addressed the individual capacity of VEGF and angiopoietins to promote the formation of mature neovessels and to identify the different types of inflammatory cells accompanying the angiogenic process over time. Sterilized polyvinyl alcohol (PVA) sponges soaked in growth factor-depleted Matrigel mixed with PBS, VEGF, Ang1, or Ang2 (200 ng/200 µl) were subcutaneously inserted into anesthetized mice. Sponges were removed at day 4, 7, 14, or 21 post-procedure for histological, immunohistological (IHC), and flow cytometry analyses. As compared to PBS-treated sponges, the three growth factors promoted the recruitment of inflammatory cells, mainly neutrophils and macrophages, and to a lesser extent, T- and B-cells. In addition, they were more potent and more rapid in the recruitment of endothelial cells (ECs) and in the formation and maturation (ensheating of smooth muscle cells around ECs) of neovessels. Thus, the autocrine/paracrine interaction among the different inflammatory cells in combination with VEGF, Ang1, or Ang2 provides a suitable microenvironment for the formation and maturation of blood vessels.

Cardiac resident nestin(+) cells participate in reparative vascularisation.

The rodent heart contains a population of nestin((+)) cells derived from the embryonic neural crest and migrate to the scar after myocardial infarction (MI). The present study tested the hypothesis that intron 2 of the nestin gene drives expression and a subpopulation of nestin((+)) cells participate in reparative vascularisation. The directed expression of the green fluorescent protein (GFP) by the second intron of the nestin gene identified GFP/nestin((+)) cells intercalated among ventricular myocytes in the heart of normal transgenic mice. Ischemic injury led to the migration of GFP((+)) cells to the scar and a subpopulation was detected in CD31/nestin((+)) endothelial cells of newly formed blood vessels. The direct contribution to reparative vascularisation provided the impetus to test the hypothesis that increasing the population of nestin((+)) cells in the infarcted heart will improve scar healing. Skin-derived cells isolated from E18 Sprague-Dawley rats grew as spheres, expressed nestin, sox2, neural crest-related transcriptional genes and a panel of peptide growth factors. Skin-derived cells transplanted in the non-infarcted left ventricle of 3-day post-MI rats migrated to the peri-infarct/infarct region and remained engrafted for 21 days. A significantly smaller infarct, increased number of small calibre blood vessels and improved ventricular function were observed in engrafted infarcted rat hearts. Thus, the second intron of the nestin gene drives expression in the mouse heart and a subpopulation of GFP/nestin((+)) cells directly participate in reparative vascularisation. Increasing the population of nestin((+)) cells via the transplantation of skin-derived cells represents a potential approach to limit ischemic damage to the heart.

Correlation between Neutrophil Extracellular Traps (NETs) Expression and Primary Graft Dysfunction Following Human Lung Transplantation.

Primary graft dysfunction (PGD) is characterized by alveolar epithelial and vascular endothelial damage and inflammation, lung edema and hypoxemia. Up to one-third of recipients develop the most severe form of PGD (Grade 3; PGD3). Animal studies suggest that neutrophils contribute to the inflammatory process through neutrophil extracellular traps (NETs) release (NETosis). NETs are composed of DNA filaments decorated with granular proteins contributing to vascular occlusion associated with PGD. The main objective was to correlate NETosis in PGD3 (n = 9) versus non-PGD3 (n = 27) recipients in an exploratory study. Clinical data and blood samples were collected from donors and recipients pre-, intra- and postoperatively (up to 72 h). Inflammatory inducers of NETs' release (IL-8, IL-6 and C-reactive protein [CRP]) and components (myeloperoxidase [MPO], MPO-DNA complexes and cell-free DNA [cfDNA]) were quantified by ELISA. When available, histology, immunohistochemistry and immunofluorescence techniques were performed on lung biopsies from donor grafts collected during the surgery to evaluate the presence of activated neutrophils and NETs. Lung biopsies from donor grafts collected during transplantation presented various degrees of vascular occlusion including neutrophils undergoing NETosis. Additionally, in recipients intra- and postoperatively, circulating inflammatory (IL-6, IL-8) and NETosis biomarkers (MPO-DNA, MPO, cfDNA) were up to 4-fold higher in PGD3 recipients compared to non-PGD3 (p = 0.041 to 0.001). In summary, perioperative elevation of NETosis biomarkers is associated with PGD3 following human lung transplantation and these biomarkers might serve to identify recipients at risk of PGD3 and initiate preventive therapies.

Colchicine reduces lung injury in experimental acute respiratory distress syndrome.

The acute respiratory distress syndrome (ARDS) is characterized by intense dysregulated inflammation leading to acute lung injury (ALI) and respiratory failure. There are no effective pharmacologic therapies for ARDS. Colchicine is a low-cost, widely available drug, effective in the treatment of inflammatory conditions. We studied the effects of colchicine pre-treatment on oleic acid-induced ARDS in rats. Rats were treated with colchicine (1 mg/kg) or placebo for three days prior to intravenous oleic acid-induced ALI (150 mg/kg). Four hours later they were studied and compared to a sham group. Colchicine reduced the area of histological lung injury by 61%, reduced lung edema, and markedly improved oxygenation by increasing PaO2/FiO2 from 66 ± 13 mmHg (mean ± SEM) to 246 ± 45 mmHg compared to 380 ± 18 mmHg in sham animals. Colchicine also reduced PaCO2 and respiratory acidosis. Lung neutrophil recruitment, assessed by myeloperoxidase immunostaining, was greatly increased after injury from 1.16 ± 0.19% to 8.86 ± 0.66% and significantly reduced by colchicine to 5.95 ± 1.13%. Increased lung NETosis was also reduced by therapy. Circulating leukocytosis after ALI was not reduced by colchicine therapy, but neutrophils reactivity and CD4 and CD8 cell surface expression on lymphocyte populations were restored. Colchicine reduces ALI and respiratory failure in experimental ARDS in relation with reduced lung neutrophil recruitment and reduced circulating leukocyte activation. This study supports the clinical development of colchicine for the prevention of ARDS in conditions causing ALI.