Hsp20 stimulates HUVEC proliferation, migration and capillary-like tube formation.

<p>(A) Recombinant human Hsp20 protein was added to HUVECs at various doses (80–2000 ng/ml) for 24 h. BSA was used as a control. Cell proliferation was determined by MTS. (B) Time-course effects of the Hsp20 protein(1000 ng/ml) on the HUVEC proliferation. (C) Representative photographs indicated the effects of recombinant human Hsp20 protein on the trans-well and tube formation of HUVECs. (D) Migration was quantified by counting cells that were moved through the membrane (Trans-well assay). (E) Tube formation was evaluated by the measurement of relative tube length. Similar results were observed in three additional, independent experiments (*, p<0.05 <i>vs.</i> Control).</p

Proposed mechanism of the Hsp20 release and its regulation of myocardial angiogenesis.

<p>Intracellular Hsp20 is released outside cardiomyocytes via exosomes, and then interacts with VEGFR2. Consequently, its downstream signaling pathways (i.e. Akt and ERK) are activated, which promote myocardial angiogenesis.</p

Hsp20 is secreted from cardiomyocytes <i>in vivo</i>.

<p>(A) The serum Hsp20 level was increased in response to <i>in vivo</i> 30 min-LAD occlusion followed by 24 h-reperfusion. Cardiac-specific overexpression of Hsp20 increased the Hsp20 concentration in the serum under basal and myocardial ischemia/reperfusion conditions. (n = 6; *, p<0.05 <i>vs.</i> WTs; #, p<0.05 <i>vs.</i> Sham groups). (B) The levels of Hsp20 in hearts from Hsp20-transgenic mice were determined by Western blot. α-Actin was used as an internal control (n = 4). (C) Myocardial ischemia/reperfusion stimulated the translocation of Hsp20 to the cardiomyocyte membrane, which was detected by fluorescence microscopy. Images are representative sections from four mice per group (green, Hsp20; red, α-Actin; Scale bar, 100 µm). (D) Quantitative data for expression of Hsp20 was evaluated using IPP 5.1 (n = 4; *, p<0.05 <i>vs.</i> WTs; #, p<0.05 <i>vs.</i> Sham groups).</p

Cardiac-specific overexpression of Hsp20 promotes myocardial angiogenesis.

<p>(A) Blood vessels were stained for CD31 (capillary density) in heart sections of WT and Hsp20 TG mice, and (B) their quantitative analysis. For quantification of positively stained vessels, five sections of each heart (n = 4 hearts per group) were analyzed by an investigator who was blinded with respect to samples. Blood vessels were detected at low magnification (×200). Images are representative sections from four mice per group (green, α-Actin; red, CD31). Scale bar, 50 µm.</p

Hsp20 is secreted from adult rat cardiomyocytes <i>via</i> exosomes, independent of the ER-Golgi protein export pathway.

<p>(A) Brefeldin A (BFA), which inhibits the classical protein transport pathway, did not block Hsp20 release into the media under either basal or hypoxia conditions (20 µM H₂O₂). However, the release of Hsp20 from cardiomyocytes was reduced by both dimethyl amiloride (DMA), an exosome inhibitor, and Methyl-β-cyclodextrin (MBC), an inhibitor of lipid raft formation via depletion of membrane cholesterol. (B) The activity of acetylcholine esterase was used to quantify the amount of exosomes present in the media after various treatments. Similar results were observed in three additional, independent experiments (*, p<0.05 vs. Basal-Control; #, p<0.05 <i>vs.</i> H₂O₂-Control).</p

Overexpression of Hsp20 in adult cardiomyocytes enhances its secretion.

<p>(A) Mild stress dose-dependently increased the amount of Hsp20 released from both Ad.GFP and Ad.Hsp20 infected myocytes, and (B) there was no difference in the release of lactate dehydrogenase (LDH), a marker of necrosis. Similar results were observed in two additional, independent experiments (*, p<0.05 vs. Ad.GFP-Control; #, p<0.05 <i>vs.</i> Ad.GFP).</p

Extracellular Hsp20 interacts with VEGFR2 and activates its downstream signaling pathways.

<p>(A) VEGFR2 coated on a plate dose-dependently captured the Hsp20 protein, whereas BSA coated did not arrest the Hsp20 protein. (B) Recombinant human Hsp20 protein significantly induced the expression of VRGFR2 in HUVECs, and co-localized with VEGFR2 in the cell surface. Images are representative sections from 20 fields per group (green, Hsp20; red, VEGFR2). Scale bar, 25 µm. (C–E) Blockade of the VEGFR2 signaling by a VEGFR2 neutralizing antibody and CBO-P11 (a VEGFR inhibitor) suppressed the HUVEC migration (C and D) and tube formation (C and E). Similar results were observed in three additional, independent experiments (*, p<0.05 vs. Control). (F and G) Immunoblots determined the levels of Akt, p-Akt, ERK and p-ERK in Hsp20-treated HUVECs. IgG or VEGFR2 antibody was pre-treated 30 min prior to the addition of Hsp20. β-actin was used as an internal control (n = 4; *, p<0.05 <i>vs.</i> Basal; #, p<0.05 <i>vs.</i> IgG+Hsp20).</p

In Vivo Delivery of Adenoviral Vector Containing Interleukin-17 Receptor A Reduces Cardiac Remodeling and Improves Myocardial Function in Viral Myocarditis Leading to Dilated Cardiomyopathy

<div><p>Th17 cells have been implicated in the pathogenesis of myocarditis. Interleukin (IL)-17A produced by Th17 cells is dispensable for viral myocarditis but essential for the progression to dilated cardiomyopathy (DCM). This study investigated whether the adenoviral transfer of the IL-17 receptor A reduces myocardial remodeling and dysfunction in viral myocarditis leading to DCM. In a mouse model of Coxsackievirus B3 (CVB3)-induced chronic myocarditis, the delivery of the adenovirus-containing IL-17 receptor A (Ad-IL17RA:Fc) reduced IL-17A production and decreased the number of Th17 cells in the spleen and heart, leading to the down-regulation of systemic TNF-α and IL-6 production. Cardiac function improved significantly in the Ad-IL17R:Fc- compared with the Ad-null-treated mice 3 months after the first CVB3 infection. Ad-IL17R:Fc reduced the left ventricle dilation and decreased the mortality in viral myocarditis, leading to DCM (56% in the Ad-IL17R:Fc versus 76% in the Ad-null group). The protective effects of Ad-IL17R-Fc on remodeling correlated with the attenuation of myocardial collagen deposition and the reduction of fibroblasts in CVB3-infected hearts, which was accompanied by the down-regulation of A distintegrin and metalloprotease with thrombospondin type 1 motifs (ADAMTS-1), Matrix metalloproteinase-2(MMP-2), and collagen subtypes I and III in the heart. Moreover, in cultured cardiac fibroblasts, IL-17A induced the expression of ADAMTS-1, MMP-2, and collagen subtypes I and III and increased the proliferation of fibroblasts. We determined that the delivery of IL-17-RA:Fc reduces cardiac remodeling, improves function, and decreases mortality in viral myocarditis leading to DCM, possibly by suppressing fibrosis. Therefore, the adenoviral transfer of the IL-17 receptor A may represent an alternative therapy for chronic viral myocarditis and its progression to DCM.</p></div

Construction of recombinant plasmid pIRES<i>-ragB-mGITRL</i> for immunization.

<p>Amplified <i>ragB</i> and <i>mGITRL</i> genes were inserted into the eukaryotic expression vectors pIRES, respectively. CMV, human cytomegalovirus immediate-early promoter; IRES, internal ribosome entry site; SV40, simian virus 40; Amp^r, ampicillin resistance gene; Neo^r, neomycin resistance gene.</p

Ad-IL-17AR:Fc administration reduces IL-17A production in the blood and the heart.

<p>(<i>A</i>) Serum IL-6 concentrations at post-infection 7 days, 2mons and 3mons in PBS-, Ad-IL-17AR:Fc- and Ad:null-treated mice (n = 5) were measured using ELISA, and the data were analyzed by a 2-factor ANOVA. ^##<i>p</i><0.01, ^#<i>p</i><0.05 vs 7 Days group, **<i>p</i><0.01, *<i>p</i><0.05 vs control group, (<i>B</i>) Serum TNF-α concentrations at post-infection 7 days, 2mons and 3mons in PBS-, AdIL-17R:Fc- and Ad:null-treated mice (n = 5) were measured using ELISA, and the data were analyzed by a 2-factor ANOVA. ^#<i>p</i><0.05 vs 7 Days group, *<i>p</i><0.05 vs control group. The data represent the mean ± SEM for 6 mice per group. (<i>C</i>)The IL-17A protein extracted from the left ventricle of the mice in the three groups at post-infection day 90, was subjected to western blotting and was probed with the indicated antibodies (n = 3). (D)The IL-17A protein level is expressed as a ratio of the GAPDH level in the same sample; the data are expressed as the mean± SEM. *<i>P</i><0.05 vs the control group. (<i>E</i>) The IL-17RA protein extracted from the left ventricle of the mice in the three groups at post-infection day 90, was subjected to western blotting and was probed with the indicated antibodies (n = 3). (<i>F</i>) The IL-17RA protein level is expressed as a ratio of the GAPDH level in the same sample; the data are expressed as the mean± SEM.</p