Endothelial Fibrosis Research Articles

Effect of Dietary Supplements with ω-3 Fatty Acids, Ascorbic Acid, and Polyphenolic Antioxidant Flavonoid on Gene Expression, Organ Failure, and Mortality in Endotoxemia-Induced Septic Rats.

Sepsis syndrome develops through enhanced secretion of pro-inflammatory cytokines and the generation of reactive oxygen species (ROS). Sepsis syndrome is characterized by vascular hyperpermeability, hypotension, multiple organ dysfunction syndrome (MODS), and increased mortality, among others. Endotoxemia-derived sepsis is an important cause of sepsis syndrome. During endotoxemia, circulating endotoxin interacts with endothelial cells (ECs), inducing detrimental effects on endothelium function. The endotoxin induces the conversion of ECs into fibroblasts, which are characterized by a massive change in the endothelial gene-expression pattern. This downregulates the endothelial markers and upregulates fibrotic proteins, mesenchymal transcription factors, and extracellular matrix proteins, producing endothelial fibrosis. Sepsis progression is modulated by the consumption of specific nutrients, including ω-3 fatty acids, ascorbic acid, and polyphenolic antioxidant flavonoids. However, the underlying mechanism is poorly described. The notion that gene expression is modulated during inflammatory conditions by nutrient consumption has been reported. However, it is not known whether nutrient consumption modulates the fibrotic endothelial gene-expression pattern during sepsis as a mechanism to decrease vascular hyperpermeability, hypotension, MODS, and mortality. Therefore, the aim of this study was to investigate the impact of the consumption of dietary ω-3 fatty acids, ascorbic acid, and polyphenolic antioxidant flavonoid supplements on the modulation of fibrotic endothelial gene-expression patterns during sepsis and to determine the effects on sepsis outcomes. Our results indicate that the consumption of supplements based on ω-3 fatty acids and polyphenolic antioxidant flavonoids was effective for improving endotoxemia outcomes through prophylactic ingestion and therapeutic usage. Thus, our findings indicated that specific nutrient consumption improves sepsis outcomes and should be considered in treatment.

Oxidized High-Density Lipoprotein Induces Endothelial Fibrosis Promoting Hyperpermeability, Hypotension, and Increased Mortality.

During systemic inflammation, reactive oxygen species (ROS) are generated in the bloodstream, producing large amounts of oxidized HDL (oxHDL). OxHDL loses the vascular protective features of native HDL, acquiring detrimental actions. Systemic inflammation promotes endothelial fibrosis, characterized by adhesion protein downregulation and fibrotic-specific gene upregulation, disrupting endothelial monolayer integrity. Severe systemic inflammatory conditions, as found in critically ill patients in the intensive care unit (ICU), exhibit endothelial hyperpermeability, hypotension, and organ hypoperfusion, promoting organ dysfunction and increased mortality. Because endothelial fibrosis disturbs the endothelium, it is proposed that it is the cellular and molecular origin of endothelial hyperpermeability and the subsequent deleterious consequences. However, whether oxHDL is involved in this process is unknown. The aim of this study was to investigate the fibrotic effect of oxHDL on the endothelium, to elucidate the underlying molecular and cellular mechanism, and to determine its effects on vascular permeability, blood pressure, and mortality. The results showed that oxHDL induces endothelial fibrosis through the LOX-1/NOX-2/ROS/NF-κB pathway, TGF-β secretion, and ALK-5/Smad activation. OxHDL-treated rats showed endothelial hyperpermeability, hypotension, and an enhanced risk of death and mortality, which was prevented using an ALK-5 inhibitor and antioxidant diet consumption. Additionally, the ICU patients showed fibrotic endothelial cells, and the resuscitation fluid volume administered correlated with the plasma oxHDL levels associated with an elevated risk of death and mortality. We conclude that oxHDL generates endothelial fibrosis, impacting blood pressure regulation and survival.

Pharmacological Targeting of Senescence with ABT-263 in Experimental Heart Failure with Preserved Ejection Fraction

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Fundação Ciência e Tecnlogia HFpEF is the most common form of heart disease in the elderly and is associated with high morbidity and mortality. Our understanding of HFpEF pathophysiology is limited and development of efficient therapies that alter the clinical course of the disease has proved greatly challenging. Although aging is a risk factor of HFpEF1, the involvement of aging hallmarks such as cell senescent and immunosenescence is unclear. Recently, a link between endothelial senescence and HFpEF development was demonstrated in mice with accelerated aging [1], endorsing anti-aging pharmacologic as potential new therapeutic alternatives for HFpEF. In fact, the senolytic (drugs that selectively promote apoptosis of senescent cells) ABT-263 has already proven efficacy in the context of cardiovascular diseases. Further studies are needed to clarify the relevance of aging and of anti-aging therapies in the context of HFpEF. Using ZSF1 obese rats (ZSF1-Ob) as model of HFpEF we showed from 18 weeks signs of immunosenescence compared to ZSF1-Ln, namely an increased frequency of circulating myeloid cells and decreased frequency of T and B cells. Concomitantly, expression of pro-inflammatory factors (IL-6, IL-1, TGF-β, TNF-α) was upregulated in peripheral blood mononuclear cells (PBMCs) of ZSF1-Ob which also displayed characteristic features of cell senescence (p21 expression, lysosomal endogenous Beta-galactosidase (SA-B-Gal) senescence-associated secretory phenotype (SASP) and up-regulation of BCL-XL. Importantly, no signs of cellular senescence (SA-B-gal, pH2AX and senescence associated pathways) were found in the main hematopoietic organs (spleen and bone marrow). Alongside, these systemic alterations, an upsurge of cellular senescence was observed in myocardium of ZSF1-Ob rats, particularly in endothelial and hematopoietic cells. Serum of ZSF1-Ob rat was able to induce activation and cellular senescence of cardiac microvascular endothelial cells, indicating that systemic circulating factors may be the upstream mechanism of cellular senescence and dysfunction in HFpEF. Analysis of HFpEF patients and a control cohort adjusted to main co-morbidities further demonstrated accumulation of senescent monocytes in HFpEF patients. In these patients, the senescence marker SA-B-Gal correlated with plasmatic brain natriuretic peptide (BNP) levels and pulmonary artery systolic pressure. Targeting aging hallmarks through the treatment of ZSF1-Ob rat with ABT-263 resulted in a reduction in circulating senescent cells, decreased systemic and local inflammation, re-established immune proportions, restore the levels of circulating BNP and attenuated myocardial remodeling, particularly endothelial dysfunction and fibrosis. Collectively these findings support that premature cellular senescence contributes to the establishment of a deleterious pro-inflammatory environment in HFpEF and that senolytic agents hold promise for the treatment of this syndrome.

The new Era of non-Steroidal Mineralocorticoid Antagonists in Treating Heart Failure in Diabetic Kidney Disease Patients.

Inappropriate activation of the mineralocorticoid receptor (MR) plays a significant role in developing hypertension, the development of cardiac pathological changes, and chronic kidney disease (CKD), where death related to cardiovascular (CV) causes is the primary competing for the outcome to the progress to end-stage renal disease (ESRD) in patients with CKD.
 Congestive heart failure (CHF) is a frequent comorbidity in patients with CKD. MR overactivation within the heart might cause coronary endothelial dysfunction, myocardial apoptosis, and reactive myocardial fibrosis and thus contribute to cardiac remodeling and its adverse consequences.
 MR antagonists (MRAs) are evidence-based therapy in symptomatic patients with heart failure and reduced ejection fraction (HFrEF).
 Finerenone, a novel, nonsteroidal, selective mineralocorticoid receptor antagonist (MRA) with anti-inflammatory and antifibrotic effects, is a promising tool in treating heart failure in patients with T2DM and CKD.
 It is felt to have higher potency and less hyperkalemia than steroidal MRAs such as spironolactone and eplerenone. It is unclear and a matter of discussion if it could be first-line therapy in this group of patients.
 Conclusion: Nonsteroidal MRAs are currently tested in clinical trials. Based on preclinical and first clinical data, these nonsteroidal MRAs might overcome the limitations of today's steroidal antagonists.

Abstract 12003: TN-C Promotes Cardiac Fibrosis and Endothelial Dysfunction in Duchenne Muscular Dystrophy

Background: Cardiac fibrosis and heart failure are major contributors to mortality in Duchenne muscular dystrophy (DMD) patients. Recently, it has been found that Tenascin C (TN-C) plays a maladaptive role in adverse left ventricular (LV) remodelling and fibrosis. Aims: To 1) elucidate the vascular dysfunction and cardiac fibrosis in link to TN-C in a mouse model of DMD and 2) explore the effect of knocking out TN-C in dystrophic mice. Methods: Male wt, mdx and mdx TN-C KO mice were used. Echocardiography was performed, and fibrosis was assessed on cardiac tissue sections. To test the vascular reactivity wire myography was used and CD31+ endothelial cells (ECs) were isolated from mouse lung tissues and characterized for oxidative stress and inflammatory marker expression. To explore further the signalling pathways contributing to cardiac fibrosis, human cardiac fibroblasts (hCFs) were treated with TN-C or TGF-β, then fibrotic markers and epigenetic regulation of p65 were assessed. Results: Cardiac fibrosis was markedly enhanced in mdx mice compared to age-matched wt animals. This was accompanied by upregulation of TN-C in cardiac tissue and plasma. In addition, vascular endothelial function was impaired in mdx mice. In line with that, endothelial cells isolated from mdx mice also showed a marked upregulation of oxidative stress and inflammatory markers. Interestingly, mdx- TN-C KO mice showed preserved vascular function as well as reduced cardiac fibrosis compared to age-matched mdx mice. hCFs treated with TN-C or TGF-β showed increased collagen production and elevated α-SMA and matrix metalloproteinase 2, 3 and 9 (MMP-2, 3 and 9) expressions. TN-C siRNA reduced the expression of collagen and α-SMA, but not MMPs. In addition, both TN-C and TGF-β promote p65/ NF-κB promoter demethylation and subsequently stimulate pro-inflammatory and pro-fibrotic signalling. These effects were reversed by applying p38 MAPK inhibitor in hCFs. Conclusions: TN-C facilitates oxidative stress and inflammation in ECs and fibroblasts, contributing to severe endothelial dysfunction and cardiac fibrosis. In addition, activation of NF-κB p65 signalling pathway may play a role in TN-C induced fibrosis. Thus, TN-C may be a critical mediator and potential target for therapy in DMD.

Abstract 12113: Deletion of Extracellular Signal-Regulated Kinases 2 Using SM22α-Cre Driver Causes Cardiac Failure and Vascular Dysfunction

Background: Extracellular signal-regulated kinases 1/2 (ERK1/2) play significant roles in proliferation, migration, and cell death. Tyrosine kinase inhibitors (TKIs) have a serious concern for cardiotoxicity and these agents target to EGFR/Ras/Raf/MEK/ERK pathway. In previous reports, SM22α-Cre drived EGFR receptor knockout mice revealed cardiac dysfunction. Hypothesis: We hypothesized if SM22α-Cre drived ERK2 contributed to the cardiac dysfunction mimicking toxicity of TKIs. Methods & Results: We generated SM22α-drived ERK2 knockout mice (SM22α-EKO) by crossing SM22α-Cre mice and ERK2-flox mice. The protein expressions of ERK2 in aorta and heart were markedly lowered in SM22α-EKO, whereas similar in brain, kidney, liver, spleen, and bone marrow. SM22α-EKO displayed lower exercise tolerance and peak oxygen consumptions. Echocardiography revealed left ventricular thickness and dilatation with decreased % fraction shortening (21±4 vs. 38±2%, p=0.0002). Pathological study showed that heart weight was twice accompanied by increases in cardiomyocyte volume, stronger cardiac fibrosis, and inflammations (Figure 1). Western blot analysis showed that the phosphorylation of Akt, p-38 MAP kinase, and STAT3 were also up-regulated in SM22α-EKO with control. Phenylephrine - induced vascular contraction was increased and acetylcholine - induced relaxation was impaired in SM22α-EKO. Finally, SM22α-EKO significantly reduced life span in the Kaplan-Meier plot (Figure 2). Conclusion: SM22α-EKO mice revealed spontaneous cardiac dysfunction with the activation of multiple signaling cascades for hypertrophy and inflammation resulting in sudden death. Deletion of ERK2 in heart and smooth muscle cells also led to hypercontraction of phenylephrine / endothelial dysfunction and perivascular cardiac fibrosis and inflammation. SM22α-EKO might be a valuable model for TKI - induced cardiotoxicity.

Abstract 10815: C-Terminal Src Kinase Inhibits Endothelial Fibrosis and is Upregulated in Early-Stage Experimental Pulmonary Arterial Hypertension

Introduction: Advanced-stage pulmonary arterial hypertension (asPAH) is characterized by endothelial dysfunction and fibrotic remodeling of pulmonary arterioles that promotes irreversible right heart failure. Thus, identifying mechanisms that regulate vascular fibrosis in early PAH (esPAH) may have translational importance. Hypothesis: We hypothesized that profibrotic molecular pathways differentiate esPAH from asPAH. Methods: To test this hypothesis, male Sprague-Dawley rats were administered one dose of monocrotaline (60 mg/kg; day 0) to induce inflammatory PAH. Cardiac catheterization and pulmonary artery endothelial cell (PAEC) transcriptomic analyses were performed on days 15 and 21 for esPAH and asPAH, respectively. Results: The esPAH profile included: right ventricular-pulmonary arterial (RV-PA) uncoupling (1.13 ± 0.05 vs. 0.90 ± 0.06, RV end-systolic elastance/PA elastance, P=0.02, N=6) and increased indexed pulmonary vascular resistance (50 ± 8 vs. 213 ± 29 mmHg*min*g -1 *mL -1 , P<0.001, N=6) without substantial elevation in PA systolic pressure (25 ± 1.2 vs. 33 ± 2.7 mmHg, P=0.02, N=6). Network medicine identified upregulation of c-terminal src kinase (Csk) as a mediator of PAEC fibrosis in esPAH but not asPAH in silico . Pulmonary endothelial expression of Csk protein by immunofluorescence correlated with vascular collagen by picrosirius red stain (r=+0.87, P=0.006, N=4). To validate these findings in vitro , human PAECs (HPAEC) were treated with an inflammatory stimulus of lipopolysaccaride (0.03 mg/mL) + interferon-γ (50 ng/mL) + interleukin-1β (50 ng/mL), or vehicle control (V). Compared to V-control, inflammation increased Csk protein and mRNA expression by 2.3- and 2.0-fold, respectively (P<0.05, N=4). Inflammation also increased HPAEC hydroxyproline, an indicator of collagen abundance, by 3.1-fold (P=1.4x10 -4 , N=4) vs. V-treated cells. Overexpression of Csk by adenovirus transfection attenuated inflammation-mediated hydroxyproline accumulation by 84% (P=1.3x10 -5 , N=3). Conclusions: These data suggest that impaired Csk may underlie HPAEC fibrosis in esPAH, which, in turn, may have potential therapeutic implications for the prevention of fibrotic vascular remodeling and PAH progression.

Biological markers of endothelial dysfunction and fibrosis, macro- and microcirculation alterations in patients with obstructive and nonobstructive coronary artery disease and diabetes mellitus

Abstract Aim of the study To analyze the association of biological markers levels of endothelial dysfunction and fibrosis with macro- and microcirculation alterations in patients with obstructive and nonobstructive coronary artery disease (CAD) and type 2 diabetes mellitus (DM). Methods 52 patients with CAD were enrolled and divided into 4 groups: 19 patients with nonobstructive CAD (1–49% stenosis) without DM (1 group: 4 men, age 64.89±7.61, body mass index (BMI) 28.14±3.69 kg/m2); 13 patients with obstructive CAD (≥50% stenosis) without DM (2 group: 9 men, age 66.92±7.02, BMI 30.4±5.91 kg/m2); 10 patients with obstructive CAD (3 group: 4 men, age 63.4±10.37, BMI 31.7±4.81kg/m2) and 10 patients with nonobstructive CAD with DM (4 group: 3 men, age 64.6±5.32, BMI 33.74±3.25 kg/m2). Patients were matched for age, sex and BMI. All patients underwent coronary angiography or coronary computed tomography angiography. Biological markers levels (E-selectin, ng/ml; tissue inhibitor of metalloproteinase 1 (TIMP-1), ng/ml) were measured using ELISA. To determine arterial damage in both macro- and microcirculation, digital reactive hyperemia photoplethysmography were performed. Endothelial function of small (Occlusion Index, OI) and large vessels (Phase Shear, PS, ms) were analyzed. Vascular remodeling of aorta (Stiffness Index, aSI, m/s) and arterioles (Reflection Index, RI, %) were studied. Results The elevation of E-selectin (1 – 21.6 [18.7; 30.4]; 2 – 31.5 [20.1; 36.9]; 3 – 42.25 [29.9; 55.3]; 4 – 42.1 [33.1; 46.5]) and TIMP-1 (1 – 416 [376; 481]; 2 – 478 [381; 539]; 3 – 534 [490; 579]; 4 – 590 [520; 782]) levels were found in all groups. Statistical analysis revealed significant differences between TIMP-1 (p1–3=0.004; p1–4=0.003) and E-Selectin (p1–3=0.013; p1–4=0.01) levels. Remodeling of large vessels was detected only in patients with obstructive CAD without DM and nonobstructive CAD with DM (2 – aSI, 9.05 [7.08; 10.58]; 3 – aSI, 8.2 [7.6; 11]). Patients in all groups had endothelial dysfunction of large vessels (PS, 1 – 5.1 [1.75; 7.75]; 2 – 6.45 [5.53; 9.03]; 3 – 7.65 [13.4; 9.5]; 4 – 4.6 [0.7; 8.1] and arterioles (IO; 1 – 1.5 [1.38; 1.78]; 2 – 1.4 [1.26; 1.53]; 3 – 1.4 [1.2; 1.7]; 4 – 1.3 [1.2; 2.0]). Structural disorders of arterioles were found in all groups, except for patients with obstructive CAD without DM (RI, 1 – 36.95 [23.4; 52.65]; 2 – 28.25 [23.35; 53.75]; 3 – 41.15 [26.5; 55]; 4 – 44.7 [20; 54.5]. The data did not show significant differences between the study groups. Conclusions The data showed that biological markers levels of endothelial dysfunction and fibrosis were increased in all groups. Significant differences revealed between the levels of E-selectin and TIMP-1 in patients with nonobstructive CAD without DM and patients with CAD and DM, regardless of the degree of stenosis. All patients had functional changes of large vessels and arterioles regardless of the severity of coronary artery atherosclerosis and presence of DM. Funding Acknowledgement Type of funding sources: None.

The Effect of Very Low Concentrations of Ethanol on Microvascular Artery and Vein Anastomosis: An Experimental Study

Objectives This study investigated the effect of very low concentrations of ethanol on artery and vein anastomosis. Also, it was aimed to determine the appropriate ethanol concentration to be used in vasospasm. Methods Rats were divided into five groups of eight rats, each group, as follows: Group i: saline; Group ii: 2.5% ethanol; Group iii: 5% ethanol; Group IV: 7.5% ethanol; and Group V: 10% ethanol. During the femoral artery and vein anastomosis, 1ml of the agent was used for irrigation in each group. Vessel diameters were measured before the anastomosis, at the fifth and 15th minutes, and the third week after the anastomosis. Histopathological samples were taken in the third week. Results In Groups ii and iii, the mean vessel diameters were found to increase 15th minute. Also observed was an increase in mean vessel diameter that continued in the third week. Although acute vasodilation was detected in Groups IV and V, arterial and venous thrombosis was observed in the third week. Intima and media thickness decreased in Group ii, while it increased in Group iii. Perivascular inflammation and fibrosis increased as the ethanol concentration increased. Conclusion 2.5% ethanol causes acute and prolonged vasodilation and does not cause endothelial cell damage, perivascular inflammation, and fibrosis. 2.5% ethanol will be a powerful alternative use in many situations that occur with vasospasm.

Identification of molecular biomarkers associated with disease progression in the testis of bulls infected with Besnoitia besnoiti

Breeding bulls infected with Besnoitia besnoiti may develop sterility during either acute or chronic infection. The aim of this study was to investigate the molecular pathogenesis of B. besnoiti infection with prognosis value in bull sterility. Accordingly, five well-characterized groups of naturally and experimentally infected males were selected for the study based on clinical signs and lesions compatible with B. besnoiti infection, serological results and parasite detection. A broad panel of molecular markers representative of endothelial activation and fibrosis was investigated and complemented with a histopathological approach that included conventional histology and immunohistochemistry. The results indicated the predominance of an intense inflammatory infiltrate composed mainly of resident and recruited circulating macrophages and to a lesser extent of CD3+ cells in infected bulls. In addition, a few biomarkers were associated with acute, chronic or subclinical bovine besnoitiosis. The testicular parenchyma showed a higher number of differentially expressed genes in natural infections (acute and chronic infections) versus scrotal skin in experimental infections (subclinical infection). In subclinical infections, most genes were downregulated except for the CCL24 and CXCL2 genes, which were upregulated. In contrast, the acute phase was mainly characterized by the upregulation of IL-1α, IL-6 and TIMP1, whereas in the chronic phase, the upregulation of ICAM and the downregulation of MMP13, PLAT and IL-1α were the most relevant findings. Macrophages could be responsible for the highest level of gene regulation in the testicular parenchyma of severely affected and sterile bulls, and all these genes could be prognostic markers of sterility.

Abstract 13662: The Arrhythmogenic Impact of Heart Failure With Preserved Ejection Fraction on Diabetics: Insights From A Nationally Representative Sample

Introduction: Heart failure with preserved ejection fraction (HFpEF) is emerging as a significant clinical concern among people with DM with recent studies showing that 45% of patients with HFpEF have DM. Sudden death accounts for around 20% of deaths in the HFpEF population, with diabetics running the highest risk. DM and HFpEF are associated with endothelial dysfunction, interstitial fibrosis, and repolarization changes that may predispose to ventricular arrhythmias (VA). However, the burden of VA in people with DM and HFpEF is not well elucidated. Hypothesis: We hypothesize that patients with DM and HFpEF have a higher prevalence of VA than patients with DM only. Methods: We queried the national inpatient sample (NIS) from 2012 to 2014 for patients age>=18years. We excluded heart failure with reduced ejection fraction and hypertrophic cardiomyopathy. Subsequently, we used ICD-9-CM codes to identify discharges associated with HFpEF, DM, VT, VF, and QTc prolongation. We divided patients into two groups: DM/HFpEF vs. DM/non-HFpEF. 1ry-outcome was VT Prevalence. 2ry-outcomes are VF prevalence, QTc prolongation, length of stay (LOS), and in-hospital mortality. We used multivariate logistic regression (MLR) to devise the propensity scores. Propensity score matching (PSM) was performed. Standardized mean difference <0.1 indicates covariate homogeneity. Results: We Identified 326545 hospitalizations among the HFpEF group and 3625130 among the non-HFpEF group. HFpEF group tended to be older, females, and had higher rates of obesity and other comorbidities and. On PSM+MLR, HFpEF group had higher Odds of VT (2.1% vs. 1.9%; OR, 1.06; 95%CI, 1.03-.10; P, <0.001) and higher LOS. Nonetheless, VF and In-hospital mortality were surprisingly higher in the non-HFpEF group. No difference in QTc prolongation between both groups. Conclusions: HFpEF was associated with higher rates of VT among patients with diabetes, although there was no change in in-hospital mortality.

Chemical proteomics reveals ligustilide targets SMAD3, inhibiting collagen synthesis in aortic endothelial cells

Atherosclerosis is a persistent inflammatory state, while vascular endothelial fibrosis is one of the primary causes of atherosclerosis development. Although ligustilide (Lig) was shown to exert obvious antiatherogenic effects in previous studies, its precise mechanism has not been deeply discussed. In this paper, we designed a Lig-derived photoaffinity labelling (PAL) probe to identify potential therapeutic targets of Lig via chemical proteomics approach. Mothers against decapentaplegic homologue 3 (SMAD3), a signal transmitter of transforming growth factor-β (TGF-β) which promotes the development of vascular fibrosis, was identified as a potential target of Lig. Lig suppressed the phosphorylation and nuclear translocation of SMAD3 by blocking the interaction between SMAD3 and TGF-β receptor 1, thereby inhibiting the collagen synthesis process. Hence, developing a novel SMAD3 inhibitor may present a promising therapeutic option for preventing vascular fibrosis.

Validity of endocan as a biomarker in systemic sclerosis: relation to pathogenesis and disease activity

BackgroundSystemic sclerosis (SSc), an autoimmune disease, has endothelial dysfunction and tissue fibrosis. The lack of its specific activity markers urges the research. Endocan is an immunomodulatory protein associated with endothelial dysfunction. We aimed to measure of the endocan level in systemic sclerosis (SSc) patients’ sera to test its relation to disease activity and treatment responses. Also, to evaluate its specificity by comparing these data with Behçet’s disease (BD) and rheumatoid arthritis (RA) patients’ data.ResultsSSc and RA patients were in activity but in the BD group; eight were active and seven were inactive. The endocan level was higher in the three groups in comparison to the controls without statistically significant differences (p value RA 0.697, SSc 0.063, and BD 0.196). A statistically significant difference in endocan levels between SSc patients with and without vascular manifestations (pulmonary hypertension and finger pitting ulcers, p < 0.0001, p = 0.0097, respectively). There were no significant correlations between endocan level and the erythrocyte sedimentation rate and C-reactive protein (positive rheumatoid factor for RA) in the three groups (p > 0.05). There were no significant correlations between endocan level and activity scores in the three groups (p > 0.05). The cut-off value of endocan was 1.3 ng/ml.ConclusionEndocan is a non-specific marker for the vascular pathogenesis in systemic sclerosis but not a good predictor for its activity.

Escleroterapia de hemangioma intra-oral: série de casos

Hemangiomas são lesões benignas de origem vascular comuns na região da cabeça e pescoço, mas dificilmente surgem em cavidade bucal. O manejo e o conhecimento das técnicas diagnósticas são fundamentais para o diagnóstico diferencial. O oleato de monoetanolamina, como agente esclerosante, promove uma reação inflamatória levando a fibrose endotelial, sendo eficaz no tratamento de lesões vasculares benignas da cavidade bucal; além das reações adversas serem de baixa intensidade e pouco relatadas na literatura; mostrando-se uma alternativa ao tratamento cirúrgico. Este trabalho teve por proposito relatar uma série de casos de pacientes submetidos à escleroterapia e discutir os resultados do tratamento de lesões vasculares bucais benignas com oleato de monoetanolamina. Quatro pacientes compareceram no serviço de Cirurgia e Traumatologia Buco-Maxilo-Facial do Hospital Universitário Oswaldo Cruz (CTBMF/HUOC/UPE) – Recife/PE com queixa de lesão oral. Após exame clínico detalhado e a utilização da vitropressão foram obtidos os diagnósticos de hemangiomas optando-se pelo tratamento à base de escleroterapia com oleato de monoetanolamina apresentando satisfatória resolução clínica. Nos casos aqui relatados, o agente esclerosante mostrou-se eficaz para o tratamento de hemangiomas em cavidade bucal.

Acacetin improves endothelial dysfunction and aortic fibrosis in insulin-resistant SHR rats by estrogen receptors

The aim of the work was to investigate the effects of acacetin on endothelial dysfunction and aortic fibrosis in insulin-resistant SHR rats and explore its mechanism. Seven-week-old male spontaneously hypertensive rats (SHR) were selected to establish a rat model of hypertension with insulin resistance induced by 10% fructose. The nuclear factor kappa B p65 (NF-κB p65) and Collagen I were observed by Immunohistochemistry. Immunofluorescence was used to observe estrogen receptor-alpha (ERα), estrogen receptor-beta (ERβ), and G protein-coupled receptor 30 (GPR30). Western blotting was used to detect interleukin (IL-1β), Arginase 2 (ARG2), Nostrin, endothelial nitric oxide synthase (eNOS), TGF-β, Smad3, ERK pathway proteins such as p-c-Raf, p-MEK1/2, p-ERK, ERK, p-P90RSK and p-MSK1. We found that acacetin did have an improvement on endothelial dysfunction and fibrosis. Meanwhile, it was also found to have a significant effect on the level of estrogen in this model by accident. Then, the experiment of uterine weight gain in mice confirmed that acacetin had a certain estrogen-like effect in vivo and played its role through the estrogen receptors pathway. In vitro experience HUVEC cells were stimulated with 30 mM/L glucose and 100 mM/L NaCl for 24 h to establish the endothelial cell injury model. HUVEC cells were treated with 1 μM/L estrogen receptors antagonist (ICI 182780) for 30 min before administration. Cell experiments showed that acacetin could reduce the apoptosis of HUVEC cells, the levels of inflammatory cytokines and the expression of TGF-β, Collagen I and Smad3 in endothelial cell injury model. After treatment with ICI 182780, the improvement of acacetin was significantly reversed. The results showed that acacetin relieved endothelial dysfunction and reduced the aortic fibrosis in insulin-resistant SHR rats by reducing the release of inflammatory factors and improving vasodilatory function through estrogen signaling pathway.

Levels of toll-like receptors 2 and 4 in serum of men with postinfarction cardiosclerosis and its relationship with antiphospholipid syndrome components

Annotation. There is a tendency for an increase of incidence of coronary heart disease (CHD) among young people, with the development of acute myocardial infarction (MI) in the absence of marked atherosclerosis and obstruction of the coronary arteries. Antiphospholipid syndrome (APS), cellular and humoral mediators of immunoinflammatory activation of endothelium are considered as factors of non-obstructive damage to the coronary vessels. New trigger factors for endothelial dysfunction, cardiac fibrosis, and myocardial hypertrophy include toll-like receptors (TLR2 and TLR4). Antiphospholipid antibodies may act as endogenous activators of toll-like receptors. The study of the association between APS components and expression of toll-like receptors in patients with CHD with postinfarction cardiosclerosis remains relevant. The aim is to study the levels of TLR2 and TLR4 and its relationship with APS components in men with postinfarction cardiosclerosis. 164 patients with stable CHD with postinfarction cardiosclerosis (100% of men, age 53.0±9.14 years) and 48 men of control group (without CHD) were examined. Levels of total antiphospholipid antibodies (aPL), antibodies to β2-glycoprotein 1 (anti-β2-GP 1) IgG and IgM, TLR2, TLR4 in serum were determined by ELISA. The studies were conducted in compliance with bioethical standards. Statistical processing of the results was performed in SPSS Statistics 22.0. Determined that men with postinfarction cardiosclerosis showed higher levels of TLR2 and TLR4 (in 1.8–2.2 times, p&lt;0.001) in serum than control group. Higher (in 1.2–1.4 times) levels of TLR2 and TLR4 were found in patients who underwent Q-MI, recurrent MI and had clinical manifestations of APS (stroke, transient ischemic attacks, livedo reticularis). An increase TLR2 and TLR4 levels was closely associated with an increase of aPL and anti-β2-GP 1 of IgG (r = | 0.31 - 0.51 |, p&lt;0.01). Thus, in men with postinfarction cardiosclerosis who had aPL and anti-β2-GP 1 IgG positivity had an unfavorable immunoinflammatory pattern, that characterized by increased expression of TLR2 and TLR4. The imbalance in TLR2 and TLR4 is more pronounced in young CHD patients and associated with Q-MI and recurrent MI.

217Metabolomic profile of patients with new onset heart failure; more microvascular dysfunction in patients with preserved ejection fraction compared to reduced ejection fraction - the PREFERS Study

Abstract Background Heart failure with preserved (HFpEF) and reduced (HFrEF) ejection fraction are both associated with metabolic derangements which may have different pathophysiological implications Purpose To identify metabolites and pathways differentially altered with the potential to differentiate HFpEF from HFrEF. Methods In the PREFERS Stockholm study (Preserved and Reduced Ejection Fraction Epidemiological Regional Study) 121 endogenous plasma metabolites were assessed by targeted mass spectrometry. Partial Least Squares Discriminant Analysis (PLS-DA) was used to identify metabolites differentially altered in new onset HF divided into HFpEF (EF ≥50%, n=46) versus HFrEF (ÈF&lt;40%, n=75) patients. Multivariable logistic regression was used to assess independent associations between HF group and selected metabolites, including sex, age and eGFR as co-variates. Results Compared to HFrEF, HFpEF patients were older; 77 vs 65 years (p&lt;0.001), more often female 54% vs 46% (p=0.004) with hypertension 60% vs 40% (p&lt;0.001) and diabetes 63% vs 37% (p=0.007), and lower NT-proBNP 720 vs 1295 ng/L (p=0.014) and eGFR 63 vs 72 mL/min/1.73 m2 (p&lt;0.001). HFpEF patients had higher levels of hydroxyproline, cysteine, symmetric dimethyl arginine, alanine, kynurenine and acylcarinitines and lower levels of cAMP, lysoPC, L-carnitine, arginine, cGMP, serine and lactate (Figure). HFpEF was independently associated with reduced levels of cGMP (OR 0.98 [95% CI: 0.97–0.99; p=0.008]), serine (0.97 [0.95–1.00; 0.047]) and cAMP (0.97 [0.94–0.99; 0.009]). Figure 1 Conclusions In new onset HF patients, HFpEF was associated with decreased cGMP, cAMP and serine indicating increased oxidative stress, impaired innate immune function and myocardial hypertrophy. HFpEF patients displayed a distinct metabolic profile reflecting increased endothelial dysfunction, hypoxia, inflammation and myocardial fibrosis pointing towards more involvement of microvascular dysfunction compared to HFrEF. Acknowledgement/Funding Science for Life Laboratory–Astra Zeneca; Mölndal, Sweden collaborative grant No. 1377

Exercise attenuates myocardial fibrosis and increases angiogenesis-related molecules in the myocardium of aged rats

Objectives The present study aimed to investigate the effect of two different exercise training protocols on myocardial fibrosis and the expression of some growth factors in aged rats. Method Twenty-four male Wistar rats were randomly assigned to high-intensity interval training (HIIT) group, continuous exercise training (CET) group, and the control group. After 6 weeks of experiment, mRNA levels of fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), adropin proteins, and myocardial fibrosis were assessed. Results HIIT and CET induced a significant increase in the FGF-2 and adropin and a decrease in the myocardial fibrosis in compared with the control group. HIIT induced a significant increase in the VEGF if compared with the control group. There was no significant difference between CET and control group. Conclusion Six weeks of HIIT and CET attenuated age-related myocardial fibrosis thereby an increase in angiogenesis-related molecules in cardiac and endothelial tissues.

Abstract 256: Cardiac Fibroblast-derived Exosomes Mediate Endothelial Dysfunction and Heart Failure

Objective: Endothelial cells (ECs) play critical role to maintain the normal heart function. Others and we have previously shown that stress-induced chronic inflammation mediates ECs dysfunction and restoration of IL10 (an anti-inflammatory cytokine) preserve ECs function, but, its mechanism is not clearly known. Here we hypothesized that activated cardiac fibroblasts-derived exosomes (FB-Exo) mediates cardiac ECs dysfunction and leads for cardiac pathology and IL10 treatment negatively regulates the FB-Exo contents and thus improve ECs function and biology. Methods: Human aortic endothelial cells (HAECs), human umbilical endothelial cells (HUVEC) and cardiac endothelial cells were cultured in EC growth media. Cells were treated with TGFβ/IL10/FB-exo. Exosomes were isolated from fibroblast condition media by ultracentrifugation and characterized by nanosight &amp; electron microscopy. Results: TGFβ treatment significantly induced ECs dysfunction as shown by Matrigel assay, real-time Q-PCR and immunostaining data. IL10 treatment significantly restored TGFβ-induced endothelial dysfunction as evident by immunostaining and western blot (CD31, Smad2/3 and αSMA) data. Furthermore, exosomes were isolated from saline/AngII treated neonatal heart fibroblast conditioned media by ultracentrifugation and characterized. We found that exosomes-derived from Ang II/TGF treated fibroblast were enriched with TGFβ and collagen-1α. Matrigel tube formation and quantitative PCR results suggest that activated fibroblasts secrete factors which triggered endothelial dysfunction (Matrigel/qPCR). Intriguingly, Ang II treated fibroblast-derived exosomes strongly promote endothelial cell differentiation to fibroblasts like cells. Conclusions: Taken together, this study demonstrates that Ang II/TGFb treated fibroblasts-derived exosomes are enriched in pro-fibrotic factors and can lead to endothelial dysfunction and promotes cardiac fibrosis in PO myocardium. Ongoing investigations using molecular approaches will provide a better understanding on the mechanistic and therapeutic aspects of IL10 and modification of fibroblast-derived exosomal content on endothelial function and cardiac fibrosis failing heart.

Endotoxemia-induced endothelial fibrosis inhibition improves hypotension, tachycardia, multiple organ dysfunction syndrome, cytokine response, oxidative stress, and survival

Sepsis syndrome is the leading cause of mortality in critically ill patients admitted to intensive care. However, current therapies for sepsis treatment are unsatisfactory, and the mortality rate is still high. The main pathological characteristics observed during sepsis syndrome and endotoxemia include hypotension, tachycardia, multiple organ dysfunction syndrome (MODS), tissue damage, and cytokine and oxidative bursts. These conditions severely decrease the survival rates of endotoxemic patients. As a consequence of endotoxemia, large amounts of endotoxin circulate in the bloodstream throughout the vascular system and interact directly with endothelial cells that cover the inner wall of blood vessels. Endothelial cells exposed to lipopolysaccharides exhibit conversion to activated fibroblasts. By means of endotoxin-induced endothelial fibrosis, endothelial cells downregulate the expression of endothelial proteins and express fibrotic and ECM markers throughout endothelial protein expression reprogramming. Although endotoxin-induced endothelial fibrosis should, in theory, be detrimental to endothelial vascular function, the role of endothelial fibrosis in sepsis syndrome or endotoxemia is not known. Therefore, we employed a rat model to investigate whether the inhibition of endotoxin-induced endothelial fibrosis protects against endotoxemia and whether this inhibition increases survival. Our results show that the inhibition of endotoxin-induced endothelial fibrosis reduced both hypotension and tachycardia. Endotoxemia-induced MODS was also decreased when endothelial fibrosis was inhibited; treated rats showed normal kidney and liver function, inhibition of muscle mass wasting and normal glycemia. Liver and kidney histology was preserved, and organ fibrosis and fibrotic protein expression were reduced. Furthermore, pro-inflammatory cytokine secretion and NOX-2-mediated oxidative stress bursts were decreased when endothelial fibrosis was inhibited. Remarkably, the risk of death associated with sepsis syndrome at early and late time points was decreased when endotoxemia-induced endothelial fibrosis was inhibited, and a significant increase in survival was observed. These results reveal a potential novel treatment strategy to protect against sepsis syndrome and endotoxemia.