Interstitial Collagen Research Articles

Clinical, radiological, and histopathological features of pulmonary post-COVID syndrome : Aform of autoimmune-mediated interstitial lung disease?

HintergrundEtwa 10 % der Patienten weisen nach einer milden COVID-19-Erkrankung anhaltende Symptome auf. Wir hatten zuvor Autoantikörper (ANA/ENA) bei Patienten mit schweren COVID-19-Verläufen erstbeschrieben.FragestellungZiel dieser Pilotstudie war die Charakterisierung der Long‑/Post-COVID-Erkrankung und der Vergleich mit einer Lungenbeteiligung bei Erkrankungen aus dem rheumatischen Formenkreis (CTD-ILD).MethodenWir rekrutierten prospektiv 33 Patienten mit anhaltender pulmonaler Symptomatik nach einer mild/moderat verlaufenen COVID-19-Erkrankung (Altersmedian 39 Jahre). Wir führten Lungenfunktionstests, Computertomografien (CT) und einen serologischen Autoantikörpernachweis durch und entnahmen bei 29 von 33 Patienten transbronchiale Lungenbiopsien.ErgebnisseDie meisten Patienten wiesen gestörten Sauerstoffpuls in der Spiroergometrie und eine leichte Lymphozytose in der bronchoalveolären Lavage auf. In der CT zeigten sich verdickte Atemwege und ein erhöhtes Low-attenuation-Volumen. Autoantikörper konnten bei 13 von 33 Patienten nachgewiesen werden (39,4 %). Histopathologisch zeigte sich interstitielle Lymphozytose mit alveolärer Fibrinausfällung und organisierender Pneumonie. Ultrastrukturelle Analysen zeigten interstitielle Kollagenablagerung.DiskussionWährend die histopathologischen Befunde der Long‑/Post-COVID-Erkrankung für sich alleine unspezifisch sind, würde die Kombination mit klinischen und radiologischen Eigenschaften die Diagnosekriterien einer „interstitiellen Pneumonie mit autoimmunen Merkmalen“ (IPAF) erfüllen. Da sich interstitielle Kollagenablagerung zeigte und die IPAF/CTD-ILD einen fibrosierenden Verlauf nehmen kann, sollte eine mögliche Persistenz von Autoantikörpern und die Entwicklung fibrotischer Lungenveränderungen bei Long‑/Post-COVID-Patienten engmaschig kontrolliert werden.

The Lung Elastin Matrix Undergoes Rapid Degradation Upon Adult Loss of Hox5 Function.

Hox genes encode transcription factors that are critical for embryonic skeletal patterning and organogenesis. The Hoxa5, Hoxb5, and Hoxc5 paralogs are expressed in the lung mesenchyme and function redundantly during embryonic lung development. Conditional loss-of-function of these genes during postnatal stages leads to severe defects in alveologenesis, specifically in the generation of the elastin network, and animals display bronchopulmonary dysplasia (BPD) or BPD-like phenotype. Here we show the surprising results that mesenchyme-specific loss of Hox5 function at adult stages leads to rapid disruption of the mature elastin matrix, alveolar enlargement, and an emphysema-like phenotype. As the elastin matrix of the lung is considered highly stable, adult disruption of the matrix was not predicted. Just 2 weeks after deletion, adult Hox5 mutant animals show significant increases in alveolar space and changes in pulmonary function, including reduced elastance and increased compliance. Examination of the extracellular matrix (ECM) of adult Tbx4rtTA; TetOCre; Hox5afafbbcc lungs demonstrates a disruption of the elastin network although the underlying fibronectin, interstitial collagen and basement membrane appear unaffected. An influx of macrophages and increased matrix metalloproteinase 12 (MMP12) are observed in the distal lung 3 days after Hox5 deletion. In culture, fibroblasts from Hox5 mutant lungs exhibit reduced adhesion. These findings establish a novel role for Hox5 transcription factors as critical regulators of lung fibroblasts at adult homeostasis.

Lingbao Huxin Pill Alleviates Apoptosis and Inflammation at Infarct Border Zone through SIRT1-Mediated FOXO1 and NF- κ B Pathways in Rat Model of Acute Myocardial Infarction

To investigate whether Lingbao Huxin Pill (LBHX) protects against acute myocardial infarction (AMI) at the infarct border zone (IBZ) of myocardial tissue by regulating apoptosis and inflammation through the sirtuin 1 (SIRT1)-mediated forkhead box protein O1 (FOXO1) and nuclear factor-κ B (NF-κ B) signaling pathways. Six-week-old Wistar rats with normal diet were randomized into the sham, the model, Betaloc (0.9 mg/kg daily), LBHX-L (0.45 mg/kg daily), LBHX-M (0.9 mg/kg daily), LBHX-H (1.8 mg/kg daily), and LBHX+EX527 (0.9 mg/kg daily) groups according to the method of random number table, 13 in each group. In this study, left anterior descending coronary artery (LADCA) ligation was performed to induce an AMI model in rats. The myocardial infarction area was examined using a 2,3,5-triphenyltetrazolium chloride solution staining assay. A TdT-mediated dUTP nick-end labeling (TUNEL) assay was conducted to assess cardiomyocyte apoptosis in the IBZ. The histopathology of myocardial tissue at the IBZ was assessed with Heidenhain, Masson and hematoxylineosin (HE) staining assays. The expression levels of tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-1 β, and intercellular adhesion molecule-1 were measured using enzyme-linked immunosorbent assays (ELISAs). The mRNA expressions of SIRT1 and FOXO1 were detected by real-time qPCR (RT-qPCR). The protein expressions of SIRT1, FOXO1, SOD2, BAX and NF- κ B p65 were detected by Western blot analysis. The ligation of the LADCA successfully induced an AMI model. The LBHX pretreatment reduced the infarct size in the AMI rats (P<0.01). The TUNEL assay revealed that LBHX inhibited cardiomyocyte apoptosis at the IBZ. Further, the histological examination showed that the LBHX pretreatment decreased the ischemic area of myocardial tissue (P<0.05), myocardial interstitial collagen deposition (P<0.05) and inflammation at the IBZ. The ELISA results indicated that LBHX decreased the serum levels of inflammatory cytokines in the AMI rats (P<0.05 or P<0.01). Furthermore, Western blot analysis revealed that the LBHX pretreatment upregulated the protein levels of SIRT1, FOXO1 and SOD2 (P<0.05) and downregulated NF- κ B p65 and BAX expressions (P<0.05). The RT-qPCR results showed that LBHX increased the SIRT1 mRNA and FOXO1 mRNA levels (P<0.05). These protective effects, including inhibiting apoptosis and alleviating inflammation in the IBZ, were partially abolished by EX527, an inhibitor of SIRT1. LBHX could protect against AMI by suppressing apoptosis and inflammation in AMI rats and the SIRT1-mediated FOXO1 and NF- κ B signaling pathways were involved in the cardioprotection effect of LBHX.

Abstract 10507: Pyridoxamine Protects Against Cardiotoxicity After Doxorubicin Chemotherapy

Introduction: Although doxorubicin (DOX) is an efficient anthracycline agent used to treat cancer, it induces cardiotoxicity and mortality in cancer survivors. Cardioprotection is inadequate. The vitamin B 6 -derivative pyridoxamine (PM) has shown to be cardioprotective in diverse cardiac diseases. Whether PM offers cardioprotection after DOX treatment is unknown. We hypothesized that PM limits cardiac impairment after DOX treatment by reducing cardiac fibrosis and inflammation. Methods: Female Sprague Dawley rats were weekly treated with 2 mg/kg DOX or saline IV for 8 weeks. At DOX treatment onset, 2 extra groups received PM (1 g/L) via the drinking water. Echocardiographic (4D) and hemodynamic parameters were assessed at week 8, together with plasma BNP. PCR analysis was performed on left ventricular tissue to evaluate fibrosis and inflammation. Data were compared using 1-way ANOVA, Kruskal-Wallis test or 2-way ANOVA with post-hoc tests as appropriate. Results: As shown in Table 1, cardiac impairment by DOX was prevented by PM. In addition, DOX significantly increased the expression of fibrosis markers, such as TGFβ (0.29 ± 0.02 vs. 0.69 ± 0.09 a.u., p&lt;0.0001), LOX (0.055 ± 0.002 vs. 0.590 ± 0.174 a.u., p&lt;0.01), collagen type 1 (0.21 ± 0.02 vs. 0.62 ± 0.11 a.u., p&lt;0.001) and interstitial collagen (4.6 ± 0.3 vs. 7.7 ± 0.6%, p&lt;0.001). DOX also increased the inflammation marker IL-6 (0.13 ± 0.03 vs. 0.73 ± 0.13 a.u., p&lt;0.01). PM treatment significantly lowered all these parameters (p&lt;0001, p&lt;0.001, p&lt;0.01, p&lt;0.01 and p&lt;0.01 respectively). Conclusions: In conclusion, our data show that DOX causes dilated cardiomyopathy with reduced ejection fraction, accompanied by cardiac fibrosis and myocarditis. As PM limits this adverse phenotype, it could be a novel cardioprotective strategy for DOX-treated cancer patients.

Resveratrol against Cardiac Fibrosis: Research Progress in Experimental Animal Models.

Cardiac fibrosis is a heterogeneous disease, which is characterized by abundant proliferation of interstitial collagen, disordered arrangement, collagen network reconstruction, increased cardiac stiffness, and decreased systolic and diastolic functions, consequently developing into cardiac insufficiency. With several factors participating in and regulating the occurrence and development of cardiac fibrosis, a complex molecular mechanism underlies the disease. Moreover, cardiac fibrosis is closely related to hypertension, myocardial infarction, viral myocarditis, atherosclerosis, and diabetes, which can lead to serious complications such as heart failure, arrhythmia, and sudden cardiac death, thus seriously threatening human life and health. Resveratrol, with the chemical name 3,5,4′-trihydroxy-trans-stilbene, is a polyphenol abundantly present in grapes and red wine. It is known to prevent the occurrence and development of cardiovascular diseases. In addition, it may resist cardiac fibrosis through a variety of growth factors, cytokines, and several cell signaling pathways, thus exerting a protective effect on the heart.

The Pathogenesis of COVID-19 Myocardial Injury: An Immunohistochemical Study of Postmortem Biopsies.

RationaleMyocardial injury associates significantly and independently with mortality in COVID-19 patients. However, the pathogenesis of myocardial injury in COVID-19 remains unclear, and cardiac involvement by SARS-CoV-2 presents a major challenge worldwide.ObjectiveThis histological and immunohistochemical study sought to clarify the pathogenesis and propose a mechanism with pathways involved in COVID-19 myocardial injury.Methods and ResultsPostmortem minimally invasive autopsies were performed in six patients who died from COVID-19, and the myocardium samples were compared to a control group (n=11). Histological analysis was performed using hematoxylin-eosin and toluidine blue staining. Immunohistochemical (IHC) staining was performed using monoclonal antibodies against targets: caspase-1, caspase-9, gasdermin-d, ICAM-1, IL-1β, IL-4, IL-6, CD163, TNF-α, TGF-β, MMP-9, type 1 and type 3 collagen. The samples were also assessed for apoptotic cells by TUNEL. Histological analysis showed severe pericardiocyte interstitial edema and higher mast cells counts per high-power field in all COVID-19 myocardium samples. The IHC analysis showed increased expression of caspase-1, ICAM-1, IL-1β, IL-6, MMP-9, TNF-α, and other markers in the hearts of COVID-19 patients. Expression of caspase-9 did not differ from the controls, while gasdermin-d expression was less. The TUNEL assay was positive in all the COVID-19 samples supporting endothelial apoptosis.ConclusionsThe pathogenesis of COVID-19 myocardial injury does not seem to relate to primary myocardiocyte involvement but to local inflammation with associated interstitial edema. We found heightened TGF-β and interstitial collagen expression in COVID-affected hearts, a potential harbinger of chronic myocardial fibrosis. These results suggest a need for continued clinical surveillance of patients for myocardial dysfunction and arrythmias after recovery from the acute phase of COVID-19.

Sickle Cell Disease with Dermatomyositis - a Rare and Complex Comorbidity

Introduction and Case Presentation - A 4 yrs old female with sickle cell disease (SCD) and intermittent asthma presented with polyarthralgia predominantly involving bilateral hip and knee joints and became non-ambulatory over a course of 2 months. She developed chronic facial swelling, and a pruritic erythematous rash involving face, extensor surface of the hand and the right knee with significant weight loss. Physical examination was significant for heliotropic rash on the eyelids, Also present were Gottron's papules and macules on both hands and right knee along with right leg tenderness, muscle weakness, wasting and decreased range of motion at bilateral hip joints along with inguinal lymphadenopathy. Investigations were significant for severe anemia with hyperactive bone marrow, metabolic acidosis, normonatremic dehydration with absence of any evidence of an ongoing infectious process. Her Anti Nucleic acid antibody (ANA) panel, Aldolase, Rheumatoid factor and Angiotensin-1-Converting Enzyme levels were unremarkable. Creatine kinase levels were near normal. Her Neopterin and LDH levels were significantly elevated. Imaging with MRI scan with multiplanar multisequence of bilateral lower extremities and Pelvis showed significant diffuse myositis and soft tissue swelling. The diagnostic criterion for Juvenile Dermatomyositis include: (1) the typical pathognomonic rashes, (2) elevated muscle enzymes, (3) symmetric proximal muscle weakness and neck flexors, (4) muscle biopsy characteristic of juvenile dermatomyositis, and (5) EMG findings characteristic of juvenile dermatomyositis. Having ruled out an infectious process a muscle biopsy was performed, which also was subjected to Electron Microscopic (EM) analysis as the patient did not have extremely high muscle enzymes, necessitating a muscle biopsy . Hematoxylin & eosin stained sections were compatible with inflammatory myopathy. Ultrastructural examination demonstrated myofiber size variability, frequent rounded atrophic fibers with myofibrillar disarray, internalized nuclei, and increased interstitial collagen deposition. Endomysial capillaries were decreased in number, but showed reactive endothelial changes. Some showed prominent endothelial tubuloreticular inclusions (Figure),characteristic of JD. In summary, absence of serologic and tissue evidence of any other inflammatory myopathy, offending pharmacotherapy, infectious disease and presence of imaging and tissue evidence (vascular injury and tubuloreticular inclusions) comprised the work up of a child with SCD with Dermatomyositis.Management- Prompt treatment with prednisone (2 mg/kg/day), methotrexate (10 mg) weekly and monthly IVIG (2gm/kg/dose) infusions was started, once diagnosis was confirmed showing a dramatic clinical response. Prednisone was slowly tapered after 1 month of treatment. She showed gradual improvement in her symptoms, the strength in proximal muscle in both upper and lower limbs improved eventually to a point where the contractures in the elbows and knees improved and she started ambulating without support. Patient's Hydroxyurea was restarted at a low dose of 15mg/kg/day and increased to 20 mg/kg/day slowly, as her systemic symptoms of Dermatomyositis subsided.Discussion- Working up a child with SCD who presents with sudden onset non weight bearing can be complicated. Vaso-occlusive bony pain crisis , Osteomyelitis , septic arthritis are common clinical scenarios, but an underlying rheumatologic illness in pediatric SCD patient mimicking a sickle cell pain crisis presents a unique diagnostic challenge one which, albeit has been reported in literature, but rarely includes electron microscopic ultrastructural examination as part of evidence and work up. This patient's relatively mild muscle enzyme elevation and Hydroxyurea therapy made the initial diagnosis more challenging. We present the first pediatric SCD patient with clinically and pathologically proven case of Dermatomyositis with EM evidence, highlighting a unique clinical scenario ,diagnostic challenges and management strategies. [Display omitted] DisclosuresJain: Octapharma: Consultancy; CSL Behring: Consultancy, Speakers Bureau; GBT: Consultancy; Blue Bird Bio: Consultancy; Takeda: Consultancy, Speakers Bureau.

Molecular programs of fibrotic change in aging human lung

Lung fibrosis is increasingly detected with aging and has been associated with poor outcomes in acute lung injury or infection. However, the molecular programs driving this pro-fibrotic evolution are unclear. Here we profile distal lung samples from healthy human donors across the lifespan. Gene expression profiling by bulk RNAseq reveals both increasing cellular senescence and pro-fibrotic pathway activation with age. Quantitation of telomere length shows progressive shortening with age, which is associated with DNA damage foci and cellular senescence. Cell type deconvolution analysis of the RNAseq data indicates a progressive loss of lung epithelial cells and an increasing proportion of fibroblasts with age. Consistent with this pro-fibrotic profile, second harmonic imaging of aged lungs demonstrates increased density of interstitial collagen as well as decreased alveolar expansion and surfactant secretion. In this work, we reveal the transcriptional and structural features of fibrosis and associated functional impairment in normal lung aging.

ХАРАКТЕРИСТИКА СОСТОЯНИЯ МИОКАРДА И ПРОФИБРОТИЧЕСКИХ ФАКТОРОВ ПРИ РАЗВИТИИ ГИПЕРТОНИЧЕСКОГО СЕРДЦА

Introduction. Myocardial damage caused by arterial hypertension is manifested by changes in myocardial geometry and mass, as well as increased content of collagen and fibrous tissue in the myocardium. Excessive accumulation of collagen in myocardium is the result of imbalance between proteolytic enzymes such as matrix metalloproteinases 1 (MMP-1) and tissue inhibitors of matrix metalloproteinase 1 (TIMMP-1), high content of transforming growth factor β1 (TFG-β1), which was found in fibroblasts and macrophages. Aim. The aim of the study was to investigate the role of MMP-1 activation, its tissue inhibitor TIMMP-1 and TGF-β1 in blood in the development of myocardial fibrosis in locomotive train workers with arterial hypertension. Material and methods. 82 patients with arterial hypertension and 20 virtually healthy control subjects were examined. The patients of the analyzed groups underwent evaluation of the data of daily blood pressure monitoring, echocardiographic examination, based on which the types of left ventricular remodeling, the content of matrix metalloproteinase 1 and its tissue inhibitor activity, the level of transforming growth factor β1 in blood serum were identified. Calculation of the volume fraction of interstitial collagen was performed. Results and discussion. The study established statistically significant signs of structural changes in the left ventricular myocardium, which primarily indicated its marked left ventricular hypertrophy against the background of arterial hypertension due to chronic pressure overload. Statistically significant increase of all levels of serum markers of collagen metabolism was revealed in patients with arterial hypertension in comparison with the control group. Patients with arterial hypertension have a restructuring of the intercellular matrix components and imbalance in the MMP-1/TIMMP-1 system characterized by a shift of the collagen balance towards fibrosis (increase of THF-β1, TIMMP-1 level, decrease of MMP-1/TIMMP-1 52 ВЕСТНИК СОВРЕМЕННОЙ КЛИНИЧЕСКОЙ МЕДИЦИНЫ 2021 Том 14, вып. 6ОРИГИНАЛЬНЫЕ ИССЛЕДОВАНИЯ ratio and increase of interstitial collagen volume fraction). Significant increase of interstitial collagen volume fraction index in the group of patients with arterial hypertension comparing to the control group also indirectly indicates high intensity of interstitial collagen genesis in myocardium of these patients. Conclusion. In arterial hypertension, serum levels of THF-β1, MMP-1, and TIMMP-1 increase, which indirectly indicates an increase in myocardial collagen content.

Postmortem Findings in Patients with COVID19 Using Multiple Organ Core Needle Biopsies

Abstract Introduction/Objective The coronavirus disease 2019 (COVID19) pandemic had caused more than 500,000 deaths in the United States. Although it mainly manifests with respiratory symptoms, postmortem examination reveals that it is more of a systemic disease affecting mutliple body organs. Methods/Case Report Postmortem needle core biopsies from multiple organs were obtained from 9 patients who died at our institution in the months of April and May of 2020 due to a confirmed SARS-CoV-2 infection by RT-PCR testing of nasopharyngeal swabs. The core biopsies from body organs included lungs (8), liver (7), kidneys (5), heart (2), spleen (2), and brain (2). Histopathological examination was performed in conjunction with a set of special and immunohistochemical stains. Electron microscopy examination was also done in 4 cases. Results (if a Case Study enter NA) The cohort consisted of 6 males and 3 females with a mean age of 70.4 years (range: 68–79). The majority had comorbidities (8/9) and presented with respiratory symptoms (9/9). The most significant postmortem findings were mainly in the lungs, including alveolar hemorrhage, hyaline membranes, fibrin thrombi, intraalveolar macrophages, type-2 pneumocyte hyperplasia, and interstitial myofibroblast reaction and collagen deposition. Immunohistochemical stains showed predominance of T-lymphocytes with a mixture of CD4 and CD8 positive cells. Examination of liver showed minimal to marked microvesicular and macrovesicular steatosis and centrilobular congestion and necrosis. Tissue from kidneys revealed mild to severe acute tubular injury. Microglial activation and Alzheimer type-II astrocytosis were noted in brain, and mild white pulp depletion was seen in the spleen. Electron microscopy showed the presence of foreign bodies suspicious for viral particles ranging in size from 52.6 to 97.9 nm in 2/4 cases. Conclusion Our findings based on postmortem core needle biopsies confirm the observation that most severely affected patients have significant pulmonary pathology. However, other organs show findings that may lead to a better understanding of this disease. Postmortem examination will continue to be an invaluable tool for studying the pathologic manifestations of COVID-19.

Study on the Mechanism of Autophagy in Rats With Radiation Induced Heart Damage at Different Time Points

It is an important mode to combine local radiotherapy with systemic targeted therapy against tumor. MTOR is an ideal choice for tumor targeted therapy, which has been identified in the cardiovascular disease. Endoplasmic reticulum stress (ERS) was involved in the process of radiation induced heart damage (RIHD) in previous study, which could trigger autophagy by inhibiting mTOR. Here we established an animal experimental model to observe the performance of RIHD at different time points, and explored the mechanism of autophagy in a rat model of RIHD.56 adult male SD rats were randomly divided into four groups (n = 14): Control group (A), Radiation group (B), Rapamycin+radiation group (C), 3-MA + radiation Group (D). Animal echocardiography was performed on the 14th and 28th days after irradiation to record left ventricular function. Levels of IL-4, TNF-α, TGF-β1 and Beclin-1 and LC3 related to autophagy were assessed by quantitative analysis of protein expression.1. On the 14th day after irradiation, radiation exposure caused a decrease in LVFS, and the decrease was recovered in D group but was not changed in C group. On the 28th day after irradiation, radiation exposure caused an increase in LVEF and LVFS, accompanied by LVAW; s thickening, and except for the decrease of LVAW; s in C group there was no change of the other parameters in C and D group compared with B group. 2. On the 14th day after the heart of the rat is irradiated, edema of myocardial cells and apoptosis increased, myocardial interstitial and perivascular inflammation cells infiltrated, and myocardial interstitial collagen deposition increased. The above-mentioned injuries were aggravated in C group and were alleviated in D group compared with B group. With time extending to 28 days after irradiation, the above-mentioned histopathological changes were progressively exacerbated. Different from changes of the 14th day, these damages were alleviated in C group and were aggravated in D group on the 28th day. 3. On the 14th day, irradiation increased the levels of autophagy-related proteins Beclin-1 and LC3, inflammatory factors IL-4, TNF-α and TGF-β1. Enhancing autophagy furtherly increased the levels of inflammatory factors IL-4, TNF-α and TGF-β1. On the 28th day, irradiation reduced the expression of Beclin-1 and LC3 but still increased the levels of IL-4, TNF-α and TGF-β1. Enhancing autophagy decreased the levels of inflammatory factors IL-4, TNF-α and TGF-β1.RIHD were progressively exacerbated with the extension of observation time. Autophagy may play a bidirectional regulatory role in radiation- induced cardiac injury, and the mechanism of action is different at different time points. On the 14th day after irradiation, activated autophagy exacerbated acute radiation heart injury to a certain extent. As time prolonged to 28 days after irradiation, down-regulated autophagy exacerbated acute radiation heart injury to a certain extent.

Potential therapeutic efficacy via avB3-integrin dependent interaction of developmental endothelial locus-1 (Del-1) after established angiotensin-II dependent hypertension

Abstract Introduction Cardiovascular diseases (CVDs) contribute to about 31% of global death. Hypertension is a major risk factor and also increases the risk of CVDs. An important regulator of systemic blood pressure in the body, the Renin Angiotensin Aldosterone system becomes overactivated during hypertension releasing a critical product, angiotensin-II (ANG-II), a known vasoconstrictor, activating the immune system leading to inflammation and as such mediating the effects of hypertension and subsequent end-organ damage due to the critical role of the innate and adaptive cells in producing pro-inflammatory cytokines such interleukin-17. Del-1, an extracellular matrix protein expressed mostly by endothelial cells is a known potent inhibitor of leukocyte recruitment. Aim We tested the hypothesis that, Del-1 via αvβ3-integrin dependent interaction protects against ANG-II induced cardiovascular remodeling after established hypertension. Methods Wild type (WT) mice were infused with 2 mg/kg/day of ANG-II for 14 days. Soluble Del-1 (Del-1-Fc) and a point mutant unable to bind to αvβ3-integrin (Del-1-RGE-Fc) was injected (50 μg/per injection) daily after 6 days of ANG-II infusion. SBP was measured using tail-cuff. Endothelial function was assessed using Mulvany myograph and quantification of inflammatory cells and IL-17 by flow cytometry. Histological staining for fibrosis, myocardial/medial hypertrophy and elastin degradation was used to evaluate both aortic and cardiac remodeling. Results After 6 days, mice infused with ANG-II showed significantly higher SBP of 140 mmHg as compared to control mice (110 mmHg). However, further progression of SBP after 6 days was blunted in infused mice treated with soluble Del-1 (ANGII+Del-1-Fc) as compared to similar increase in SBP in both infused mice treated with the point mutant (ANGII+Del-1-RGE-Fc) and non-treated infused mice (ANGII+Fc) leading to a difference of ∼20 mmHg. This effect was due to, the well protected endothelium dependent relaxation and reduced wall tension of the aorta in ANGII+Del-1-Fc mice. Further infiltration of CD45+ leukocytes, CD45+/IL-17A+ and TCRβ+T cells was significantly reduced in the aorta and heart of ANGII+Del-1-Fc mice as compared to both ANGII+Del-1-RGE-Fc and ANGII+Fc mice. However, the aorta and heart of ANGII+Del-1-Fc mice showed increased levels of anti-inflammatory regulatory T-cells (CD25+FOXP3+Treg). Also, aortic medial thickness, adventitial collagen and elastin degradation was decreased significantly in ANGII+Del-1-Fc mice. Additionally, in comparison to both ANGII+Del-1-RGE-Fc and ANGII+Fc mice, the hearts of ANGII+Del-1-Fc mice had significantly less interstitial and pericoronary collagen, smaller cardiomyocyte area and subsequently higher lumen-to-wall ratio. Conclusion Our study reveals the cardiovascular protective effects of Del-1 mediated via its association with αvβ3-integrin after established hypertension which could serve as potential therapeutic target. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): German Research Foundation (DFG)National Institutes of Health (NIH)

Effects of omega 3 on doxorubicin-induced cardiotoxicity in an experimental rabbit model

To evaluate the effects of the use of omega 3 at different doses, following the response through stereological and morphometric evaluations in doxorubicin-induced acute cardiotoxicity in an experimental rabbit model. Thirty healthy adult New Zealand rabbits were divided into six groups with different doses of omega 3 treatment, GT125, GT500 and GT 2000, receiving doses of 125mg / kg, 500mg / kg and 2000mg / kg, respectively. The induction of acute cardiotoxicity with doxorubicin was treated for six days prior to intravenous administration of 10mg / kg single dose. Then, the animals were euthanized and myocardial samples were collected for stereological and morphometric evaluations. The stereological evaluation showed an increase in the transverse area of ​​cardiomocytes between the SHAM and GT2000 groups and an increase in the interstitial collagen volume density between the SHAM and GT500 and SHAM and GT2000 groups. The omega 3 500mg/kg dose has the potential to attenuate the fibrotic effects of doxorubicin use and the use of a single dose of 10mg/kg doxorubicin can be used as an acute dilated cardiomyopathy induction protocol in an experimental model using rabbits.

The Dose-Dependent Effects of Spironolactone on TGF-β1 Expression and the Vulnerability to Atrial Fibrillation in Spontaneously Hypertensive Rats.

Objective This study tends to assess the dose-dependent effects of spironolactone on TGF-β1 expression, atrial fibrosis, and the vulnerability to atrial fibrillation in spontaneously hypertensive rats (SHRs) and tries to clarify the association of atrial fibrosis with the vulnerability to atrial fibrillation. Methods Forty 20-week-old male SHRs were randomly divided into 4 groups (10 rats per group): 3 spironolactone groups were lower-dose group (10 mg·kg−1·d−1, dissolved in 2 ml saline solution, group SL), medium-dose group (40 mg·kg−1·d−1, dissolved in 2 ml saline solution, group SM), higher-dose group (80 mg·kg−1·d−1, dissolved in 2 ml saline solution, group SH) and one hypertension group (2 ml saline solution for stomach gavage, group H). Ten matched homologous WKY rats were set as the control group (group C). After 7 weeks of gavage, a multiple electroconductive physiological recorder was used to detect atrial electrical parameters, including P-wave duration, PR interval, and atrial effective refractory period (AERP), the inducibility, and duration of atrial fibrillation. HE staining was used to determine myocardial cell size. Masson staining was used to detect the deposition of the interstitial collagen fibers in atrial muscle. The expression of TGF-β1 was detected by immunohistochemistry and western blot. Results Compared with group C, the myocardial cell size, atrial fibrosis, TGF-β1 expression, P-wave duration, PR interval, AERP, inducibility, and duration of atrial fibrillation in group H were conspicuously increased (p < 0.05); compared with group H, there was no significant difference in the myocardial cell size, atrial fibrosis, TGF-β1 expression, and electrophysiological indexes in group SH upon spironolactone intervention (p > 0.05); compared with group H, the myocardial cell size, atrial fibrosis, the expression of TGF-β1, P-wave duration, PR interval, the inducibility, and duration of atrial fibrillation in the group SL and group SM were all decreased (p < 0.05); compared with group SM, the effect in the group SL was more prominent (p < 0.01). Conclusion Hypertension can lead to cardiomyocyte hypertrophy, deposition of interstitial fibrosis in myocardial tissue, and an increase in the vulnerability to atrial fibrillation. Spironolactone showed a certain dose-dependent effect in SHRs. Lower-dose spironolactone was superior to higher-dose spironolactone in the aspect of reducing hypertensive atrial fibrosis and TGF-β1 expression, as well as preventing the occurrence of atrial fibrillation.

Cardioprotective effects of the proline-rich oligopeptide Bj-PRO-7a in spontaneously hypertensive rats.

The proline-rich oligopeptide from Bothrops jararaca snake venom, Bj-PRO-7a, promotes acute effects in blood pressure in hypertensive animals. However, the cardiac effects of this heptapeptide are completely unknown. Thus, we sought to evaluate whether the Bj-PRO-7a could protect against cardiac remodelling in spontaneously hypertensive rats (SHR). SHR were treated with Bj-PRO-7a (71nmol/kg/day, s.c.) or saline for 28days. Wistar rats were used as control. Systolic blood pressure (SBP) and heart rate (HR) were measured by tail-cuff plethysmography. Cardiomyocyte diameter and interstitial and perivascular fibrosis of the left ventricle (LV) were evaluated using Picrosirius staining. Immunofluorescence was used to detect collagen I and III. Fibroblast proliferation was assessed by immunohistochemistry to detect proliferating cell nuclear antigen (PCNA). Protein expression was assessed by western blot. The superoxide dismutase and catalase activities and the concentration of lipid peroxidation products were evaluated in the LV. The SBP and HR were not different between treated and non-treated SHR at the end of the treatment. However, Bj-PRO-7a attenuated the cardiomyocyte hypertrophy, deposition of interstitial and perivascular fibrosis and collagen I, and positive PCNA-labelled fibroblasts. This peptide also reduced the increased levels of TBARS, expression and activity of catalase, and activity of SOD in LV from SHR. Also, the Bj-PRO-7a increased the expression of metalloproteinases-2 in SHR hearts. These findings demonstrate that the Bj-PRO-7a reduced the pathological cardiac remodelling in a pressure-independent manner in hypertensive rats through mechanisms mediated by oxidative stress regulation.

Abstract P326: Nicotinamide Riboside Kinase-2 Deficiency Promotes Pressure Overload- Induced Dilated Cardiomyopathy And Heart Failure

Dilated cardiomyopathy (DCM) is one of the leading causes of heart failure with a poor prognosis. Nicotinamide riboside kinase-2 (NRK-2), a muscle-specific β1 integrin-binding protein, is predominantly expressed in skeletal muscle and upregulates in several heart failure models. Emerging pieces of evidence suggest that NRK-2 plays a key role in cardiac pathogenesis, however, its role in chronic pressure overload (PO)-induced cardiac remodeling is largely unknown. To investigate the potential role of NRK-2 in PO-induced DCM and delineate the underlying molecular mechanisms, NRK2 knockout (KO) and littermate control mice were subjected to trans-aortic constriction (TAC) or sham surgeries and cardiac function was assessed by serial M-mode echocardiography. A mild cardiac contractile dysfunction was observed in the KOs at the early adaptive phase of remodeling followed by a significant LV chamber dilatation [LVIDd(mm); 5.32±0.36 vs. 4.75±0.39, P =0.001] and functional deterioration [LVEF(%); 20.53±8.81vs. 31.2±7.0, P =0.003] during the maladaptive cardiac remodeling phase (6 week). Consistently, NRK2 KO hearts displayed increased cardiac hypertrophy and heart failure reflected by morphometric parameters as well as increased fetal genes ANP and BNP expressions. Histological assessment revealed an extensive left ventricular (LV) chamber dilatation accompanied by elevated cardiomyopathy and fibrosis in the KO hearts post-TAC. In a gain-of-function model, NRK-2 overexpressing AC16 cardiomyocytes displayed significantly attenuated fetal genes expression and, NRK-2 further suppressed the fetal gene expression when challenged with angiotensin II. Consistently, NRK-2 overexpression attenuated angiotensin II-induced cardiomyocyte death. Mechanistically, NRK-2 was identified as a critical regulator of JNK MAP kinase and NRK-2 overexpression markedly suppressed the angiotensin II-induced JNK activation. Overall, our results demonstrate that NRK-2 regulates dilatative cardiac remodeling and, genetic ablation exacerbates dilated cardiomyopathy, interstitial collagen deposition, and cardiac dysfunction post-TAC due, in part, to increased JNK activation.

Abstract MP25: Defining The Role Of Renin Lineage Cells During Regeneration After Release Of Partial Ureteral Obstruction In Neonatal Mice.

Our previous study on a partial unilateral ureteral obstruction (pUUO) model in neonatal mice showed that the release of obstruction halts the progression of kidney damage and leads to a remarkable repair of the kidney with improvement in renal blood flow. In the current study, we aim to understand the role of mural cells of the renin lineage during kidney damage and repair in the neonatal pUUO model. Our results show a marked increase in renin-positive areas in kidneys obstructed for 3W (Sham-3W: 0.70±0.10%, n=3; Obstructed-3W: 1.82±0.43%, n=3). However, relief of obstruction at 1W restored the renin-positive areas to sham levels (Post-release-2W: 0.70±0.09%; n=3). Lineage tracing using Ren1 d Cre;mTmG mice revealed a significant increase in GFP+ cells in the obstructed kidneys, with a decrease post-release. To understand further the dynamic changes in cells of renin lineage due to obstruction, we ablated the renin cells using DTA (Diphtheria toxin subunit A). We crossed the DTA fl/fl mice with Ren1 d -DTA het ;Ren1 d Cre;mTmG mice and performed pUUO in the resultant pups with DTA in the renin cells (DTA+). DTA+ animals showed thinning of the renal vasculature and a 90% reduction in renin-positive area compared to controls [Control: 0.70±0.10% (n=3); DTA+: 0.06±0.03% (n=3)]. In addition, there was no significant increase in the renin-positive area post-obstruction [Sham-3W: 0.06±0.04% (n=3); Obstructed-3W: 0.12±0.05% (n=4); Post-release-2W: 0.08±0.03% (n=4)]. These results indicate that ablation of renin cells abolished the obstruction-mediated surge in the renin expression. However, measurement of interstitial collagen positive area indicated that despite the absence of renin cells, the fibrotic damage due to obstruction recovered remarkably post-release [Collagen positive area: Sham-3W: 3.38±0.67% (n=3) Obstructed-3W: 62.98±31.50% (n=3); Post-release-2W: 10.93±5.46% (n=4)]. Similarly, vascular damage induced by persistent obstruction and recovery following the relief of obstruction was similar between the DTA+ and non-DTA animals. Our results imply that though the renin and renin lineage cells increase in obstructed kidneys, ablation of renin cells does not affect the regeneration capacity of kidneys following the relief of obstruction.

Collagen Type III and VI Remodeling Biomarkers Are Associated with Kidney Fibrosis in Lupus Nephritis.

Lupus nephritis (LN) occurs in <40% of patients with SLE. Reliable biomarkers of kidney damage are needed to identify patients with SLE at risk of developing LN to improve screening, treat the disease earlier, and halt progression to kidney failure. Novel biomarkers of extracellular matrix remodeling were evaluated as markers of kidney fibrosis and disease activity in patients with LN. Biomarkers of the interstitial collagen type III (PRO-C3) and type VI (PRO-C6) formation and of collagen type III (C3M) degradation were evaluated in the serum and urine of 40 patients with LN, 20 patients with SLE but without LN, 20 healthy controls, and ten biopsy controls (histologic kidney inflammation/damage without SLE). Their association with histologic markers of interstitial fibrosis and tubular atrophy, with inflammatory cell infiltration and with disease activity and chronicity in the patients with LN was assessed. Despite PRO-C3 (serum) and PRO-C6 (serum and urine) being significantly elevated in patients with LN compared with healthy controls, the markers did not differentiate patients with LN from those with SLE. C3M (urine) levels were not different in LN compared with the other groups. C3M (urine) strongly correlated and PRO-C6 (serum and urine) inversely correlated with kidney function (eGFR). The biomarkers of interstitial collagen turnover PRO-C6 (serum) and C3M (urine) correlated with histologic markers of interstitial fibrosis, tubular atrophy, and monocyte infiltration. Noninvasive collagen turnover biomarkers are promising tools to identify patients with SLE with kidney histologic modifications.

Abstract P173: The Impact Of Cigarette Smoking-oral Contraceptive Combination On Cardiac Remodeling In Premenopausal Female Mice

Introduction: Tobacco smoke is a major risk factor for coronary artery diseases (CAD) and chronic tobacco smoking (CS) increase the risk of CAD by 2 to 4 folds. CS effect on cardiac homeostasis has never been evaluated in premenopausal females taking oral contraceptive (OC). This study investigates CS effect on cardiac remodeling in female mice in the presence or absence of Ethinyl Estradiol (EE). Methods: Blood pressure and echocardiography were recorded for female C57BL/6J mice on EE, EE-CS, vehicle, and vehicle-CS at baseline and after 8-weeks of exposure. Cardiac inflammatory cytokines and oxidative stress markers were evaluated by real time PCR and western blots. Interstitial collagen deposition was assessed by Masson Trichrome staining. Results: Eight weeks of EE-CS treated mice showed no effect on BP but significant adverse structural and functional cardiac effects, represented by increased systolic (0.981 ± 0.035 N=11 (p&lt;0.0332)) and diastolic (1.450 ± 0.037 N=11 (p&lt;0.0002)) areas when compared to the vehicle and EE groups, along with increased systolic (1.981 ± 0.109 N=11 (p&lt;0.033)) and diastolic ( 3.911 ± 0.159 N=11 (p&lt;0.033)) volumes when compared to all groups. Functional changes were accompanied with a decreased fractional shortening (9.194 ± 1.006 N=11 (p&lt;0.033)) when compared to vehicle and EE groups with no changes in ejection fraction parameter. Moreover EE-CS treatment significantly increased NOX-4 expression (2.297 ± 0.643 N=5 (p&lt;0.033)) when compared to EE treatment alone, along with an increased pro-inflammatory profile including IL-1β (0.750 ±0.171N=5 (p&lt;0.033)) and IL-4 (4.110 ± 0.623 N= 5 (p&lt;0.002)) when compared to all groups, and IL-6 (3.045±0.910 N=5 (p&lt;0.002)) and IL-13 (2.686 ± 0.648 N=5 (p&lt;0.033)) when compared to vehicle-CS group. Morphologically, EE-CS exhibited a significant increase in interstitial fibrosis (1.186± 0.020 N=5, 3.2285 ± 0.683 N=5, (p&lt;0.033)) when compared to the vehicle-CS group. Conclusion: This study provides clear evidence that CS-exposed premenopausal female mice on OC regimen exhibited significant adverse cardiac events that could be classified under cardiac dysfunction with preserved ejection fraction. Additional experiments are warranted to further elaborate on these findings.

Direct Blood Pressure-Independent Anti-Fibrotic Effects by the Selective Nonsteroidal Mineralocorticoid Receptor Antagonist Finerenone in Progressive Models of Kidney Fibrosis

Introduction: The nonsteroidal mineralocorticoid receptor (MR) antagonist finerenone and sodium-glucose cotransporter-2 (SGLT2) inhibitors have demonstrated clinical benefits in chronic kidney disease patients with type 2 diabetes. Precise molecular mechanisms responsible for these benefits are incompletely understood. Here, we investigated potential direct anti-fibrotic effects and mechanisms of nonsteroidal MR antagonism by finerenone or SGLT2 inhibition by empagliflozin in 2 relevant mouse kidney fibrosis models: unilateral ureter obstruction and sub-chronic ischemia reperfusion injury. Methods: Kidney fibrosis was induced in mice via unilateral ureteral obstruction or ischemia. In a series of experiments, mice were treated orally with the MR antagonist finerenone (3 or 10 mg/kg), the SGLT2 inhibitor empagliflozin (10 or 30 mg/kg), or in a direct comparison of both drugs. Interstitial myofibroblast accumulation was quantified via alpha-smooth muscle actin and interstitial collagen deposition via Sirius Red/Fast Green staining in both models. Secondary analyses included the assessment of inflammatory cells, kidney mRNA expression of fibrotic markers as well as functional parameters (serum creatinine and albuminuria) in the ischemic model. Blood pressure was measured via telemetry in healthy conscious compound-treated animals. Results: Finerenone dose-dependently decreased pathological myofibroblast accumulation and collagen deposition with no effects on systemic blood pressure and inflammatory markers in the tested dose range. Reduced kidney fibrosis was paralleled by reduced kidney plasminogen activator inhibitor-1 (PAI-1) and naked cuticle 2 (NKD2) expression in finerenone-treated mice. In contrast, treatment with empagliflozin strongly increased urinary glucose excretion in both models and reduced ischemia-induced albuminuria but had no effects on kidney myofibroblasts or collagen deposition. Discussion/Conclusion: Finerenone has direct anti-fibrotic properties resulting in reduced myofibroblast and collagen deposition accompanied by a reduction in renal PAI-1 and NKD2 expression in mouse models of progressive kidney fibrosis at blood pressure-independent dosages.