Reduction In Capillary Density Research Articles

A Blood Supply Pathophysiological Microcirculatory Mechanism for Long COVID.

The term "Long COVID" is commonly used to describe persisting symptoms after acute COVID-19. Until now, proposed mechanisms for the explanation of Long COVID have not related quantitative measurements to basic laws. In this work, a common framework for the Long COVID pathophysiological mechanism is presented, based on the blood supply deprivation and the flow diffusion equation. Case-control studies with statistically significant differences between cases (post-COVID patients) and controls, from multiple tissues and geographical areas, were gathered and tabulated. Microvascular loss (ML) was quantified by vessel density reduction (VDR), foveal avascular zone enlargement (FAZE), capillary density reduction (CDR), and percentage of perfused vessel reduction (PPVR). Both ML and hemodynamic decrease (HD) were incorporated in the tissue blood supply reduction (SR) estimation. ML data were found from 763 post-COVID patients with an average VDR, FAZE, CDR, and PPVR of 16%, 31%, 14%, and 21%, respectively. The average HD from 72 post-COVID patients was 37%. The estimated SR for multiple tissues with data from 634 post-COVID patients reached a sizeable 47%. This large SR creates conditions of lower mass diffusion rates, hypoxia, and undernutrition, which at a multi-tissue level, for a long time, can explain the wide variety of the Long COVID symptoms. Disruption of peripheral tissue blood supply by the contribution of both ML and HD is proposed here to be the principal cause of the mechanism leading to Long COVID symptoms.

Nailfold capillaroscopy abnormalities and pulmonary hypertension in mixed connective tissue disease and systemic sclerosis patients.

Pulmonary arterial hypertension (PAH) represents an important vascular complication of mixed connective tissue disease (MCTD) and systemic sclerosis (SSc). Microvascular involvement in these diseases can be investigated by means of nailfold capillaroscopy (NFC). Microvascular involvement detected in the nailfold bed is the mirror of the microvascular damage occurring in the entire body, further indicating the involvement of the target organs. The aim of this study was to evaluate the microvascular involvement in MCTD patients with or without PAH, compared to that found in SSc patients with or without PAH. This cross-sectional study was performed in the Department of Internal Medicine and Department of Rheumatology, Timișoara, Romania, during the time period between January 2017 and December 2022, on a group of 26 patients with MCTD and 26 SSc patients. Antinuclear antibodies, anti-U1-RNP, anti-Scl 70, anti-centromere, anti-cardiolipin antibodies (aCL) (IgM, IgG), anti-β2-glycoprotein I (aβ2GPI) (IgM, IgG) antibodies, and lupus anticoagulant (LAC) were determined in both the groups. PAH was evaluated through cardiac ultrasonography, determining the sPAP (systolic pulmonary artery pressure). Nailfold capillaroscopy was performed using a USB Digital Microscope and 2.0-megapixel digital camera recording capillaries density, giant capillaries, enlarged capillaries, capillaries hemorrhages, avascular areas, ramified/bushy capillaries scores. Data were recorded and presented as mean ± standard deviation. Statistical analyses were performed using the Student's t-test, ANOVA test, and Pearson's correlation. Differences were considered statistically significant if p-value < 0.05. Among the MCTD patients, PAH was identified in 12 patients (46.15%), while among the SSc patients PAH was identified in 14 patients (53.84%). Development of PAH in MCTD patients was associated with lower capillaries density (p-value < 0.00001), higher scores of giant capillaries, ramified/bushy capillaries, and capillary hemorrhages (p-value < 0.00001, for each of them). Anti-U1-RNP, aCL, aβ2GPI antibodies and LAC were also found to be involved in PAH-associated MCTD development. Unlike MCTD patients, SSc patients with PAH presented with lower capillaries density and ramified/bushy capillaries scores (p-value < 0.05). The MCTD patients who presented significant NFC abnormalities (especially active and late scleroderma-like capillaroscopic pattern) are prone to PAH development. Capillary density reduction is the most important factor associated with the occurrence of PAH. Differences in NFC findings (especially capillary density and ramified/bushy capillaries) were detected among patients with MCTD and SSc having PAH.

Myocardial perfusion in cardiac amyloidosis.

Cardiac involvement is the main driver of clinical outcomes in systemic amyloidosis and preliminary studies support the hypothesis that myocardial ischaemia contributes to cellular damage. The aims of this study were to assess the presence and mechanisms of myocardial ischaemia using cardiovascular magnetic resonance (CMR) with multiparametric mapping and histopathological assessment. Ninety-three patients with cardiac amyloidosis (CA) (light-chain amyloidosis n = 42, transthyretin amyloidosis n = 51) and 97 without CA (three-vessel coronary disease [3VD] n = 47, unobstructed coronary arteries n = 26, healthy volunteers [HV] n = 24) underwent quantitative stress perfusion CMR with myocardial blood flow (MBF) mapping. Twenty-four myocardial biopsies and three explanted hearts with CA were analysed histopathologically. Stress MBF was severely reduced in patients with CA with lower values than patients with 3VD, unobstructed coronary arteries and HV (CA: 1.04 ± 0.51 ml/min/g, 3VD: 1.35 ± 0.50 ml/min/g, unobstructed coronary arteries: 2.92 ± 0.52 ml/min/g, HV: 2.91 ± 0.73 ml/min/g; CA vs. 3VD p = 0.011, CA vs. unobstructed coronary arteries p < 0.001, CA vs. HV p < 0.001). Myocardial perfusion abnormalities correlated with amyloid burden, systolic and diastolic function, structural parameters and blood biomarkers (p < 0.05). Biopsies demonstrated abnormal vascular endothelial growth factor staining in cardiomyocytes and endothelial cells, which may be related to hypoxia conditions. Amyloid infiltration in intramural arteries was associated with severe lumen reduction and severe reduction in capillary density. Cardiac amyloidosis is associated with severe inducible myocardial ischaemia demonstrable by histology and CMR stress perfusion mapping. Histological evaluation indicates a complex pathophysiology, where in addition to systolic and diastolic dysfunction, amyloid infiltration of the epicardial arteries and disruption and rarefaction of the capillaries play a role in contributing to myocardial ischaemia.

Microvascular status in juvenile Sjögren’s disease: the first nailfold videocapillaroscopy investigation

IntroductionJuvenile Sjögren’s disease (jSjD) is a rare autoimmune disease characterized by exocrine gland involvement and systemic manifestations, including small vessel vasculitis and Raynaud’s phenomenon (RP). We aimed to investigate the microvascular status in jSjD patients by nailfold videocapillaroscopy (NVC) and the potential correlations with clinical and serological features.MethodsClinical data from thirteen consecutive jSjD patients (11 females and 2 males), with a mean age of 16 ± 4 years, diagnosed before 16 years of age (mean age at diagnosis 12 ± 3) according to the 2016 American College of Rheumatology/EULAR criteria for adult SjD, were collected including age- and sex-matched healthy controls (HCs). Clinical, laboratory, and instrumental data were collected, together with NVC examination. Non-specific and specific NVC parameters were investigated, such as capillary density, capillary dilations, giant capillaries, microhaemorrhages and abnormal shapes. Associations between NVC findings and clinical/serological features were explored and analysed using parametrical and non-parametrical tests.ResultsCapillary density reduction correlated significantly with articular involvement (arthralgias) (p = 0.024). Microhaemorrhages correlated with lower C3 levels (p = 0.034). No specific NVC pattern for jSjD was identified, whereas abnormal capillary shapes were significantly higher in jSjD patients than HCs (p = 0.005). NVC abnormalities were not associated with SjD-specific instrumental tests (biopsy, imaging, Schirmer’s test). RP was present in 8% of jSjD patients.ConclusionsThe reduction of capillary density, as well as microhaemorrhages at NVC analysis, are significantly associated with some clinical aspects like articular involvement and serum biomarkers (C3 reduction). The NVC is suggested as safe and further analysis in jSjD patients.Key Points• Juvenile Sjögren’s disease (jSjD) exhibits a higher rate of abnormal shapes at nailfold videocapillaroscopy (NVC) compared to matched healthy controls.• Articular involvement in jSjD is significantly associated with a lower capillary number at NVC.• Microhaemorrhages on NVC are associated with lower C3 levels, prompting this finding as a putative novel negative biomarker/prognostic factor for jSjD.

Abstract 13882: Myocardial Hypoperfusion in Severe Aortic Stenosis is Reversed by Aortic Valve Replacement

Background: Myocardial hypoperfusion in severe aortic stenosis (AS) without obstructive coronary disease may be secondary to hemodynamic influences (and therefore readily reversible by aortic valve replacement[AVR]) or structural alterations in the left ventricle (LV) such as increased mass and reduction in capillary density. Aim: To evaluate the early effect of AVR on myocardial blood flow (MBF) in patients with severe symptomatic AS without flow-limiting coronary artery disease. Methods: Prospective case-control study of severe symptomatic AS without obstructive coronary disease referred for surgical or transcatheter AVR and propensity matched to a control cohort with unobstructed coronary arteries. Patients underwent adenosine stress cardiovascular magnetic resonance at baseline and 8-weeks (median) post-operatively. Histology from 9 myocardial septal biopsies taken at AVR were compared against necropsy controls (n=5). Results: 52 patients were included; 26 with severe AS (median [IQR] age 71[66-75], 73% male) and 26 age, sex and co-morbidity matched controls. In AS, peak velocity(Vmax) was 4.3m/s[4.1-4.5] and AVA 0.8cm 2 [0.6-0.9]. AS patients had greater LV mass and late gadolinium enhancement. AS patients had lower stress MBF than controls (1.63ml/g/min[1.38-2.08] vs 2.13[1.76-2.52], p=0.003) which increased to 1.97ml/g/min after AVR (p=0.7 post-AVR vs. Controls). This was driven by a 40% increase in subendocardial MBF (1.26ml/g/min[1.18-1.78] to 1.77[1.62-2.32]). Capillary density was reduced almost threefold in AS (495/mm 2 [407-686] vs 1376[1208-1579], p&lt;0.001). Conclusions: Severe AS results in subendocardial hypoperfusion which is rapidly improved after AVR. There is a threefold reduction in capillary density, however factors related to elevated afterload (which is reduced by AVR) are the most influential.

Heart failure, female sex and atrial fibrillation are the main drivers of atrial cardiomyopathy in cardiac surgery patients: results from the CATCH ME consortium

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): CATCH ME: Characterizing Atrial fibrillation by Translating its Causes into Health Modifiers in the Elderly Background Atrial cardiomyopathy is emerging as independent prognostic factor in cardiovascular disease. Fibrotic remodeling, cardiomyocyte hypertrophy and a reduction in capillary density are histological hallmarks of atrial cardiomyopathy. The contribution of etiological factors to atrial cardiomyopathy has not been robustly quantified. Purpose To quantify the relation between histological features of atrial cardiomyopathy and the clinical profile of patients. Methods We examined left (LA, n=91) and right (RA, n=75) atrial appendages sampled from a European cohort of patients undergoing cardiac surgery. Quantification of histological cardiomyopathy features was performed a recently developed and validated imaging analysis algorithm following myocardial triple staining with wheat germ agglutinin (WGA), CD31 and vimentin. The contributions of AF, heart failure (HF), sex, age, and 4 principal components accounting for confounding clinical characteristics to over-all and endomysial fibrosis were determined in a multivariate model. K-means clustering of 6 atrial histological features was performed to identify different types of remodeling. Results LA samples showed less fibrosis (p&amp;lt;0.001), a higher capillary density (p&amp;lt;0.05), a smaller capillary size (p&amp;lt;0.05), and larger cardiomyocytes (p&amp;lt;0.01) than RA samples. In both LA and RA, persistent AF was associated with endomysial fibrosis, while over-all fibrosis was not. Men had larger cardiomyocytes (LAA), while women had a higher degree of endomysial fibrosis (LA). Heart failure patients had more endomysial and over-all fibrosis (LA), a higher capillary density (LA) and capillary size (LA/RA). Samples clustered into 2 distinct clusters. One cluster showed fibrotic cardiomyopathy, with more endomysial (p&amp;lt;0.001) and overall fibrosis (p&amp;lt;0.001), more fibroblasts (p&amp;lt;0,001) and a higher capillary density (p&amp;lt;0.001). The other cluster showed hypertrophic cardiomyopathy, with hypertrophic cardiomyocytes (p&amp;lt;0.001). Patients with fibrotic cardiomyopathy were more often female (LA and RA, p&amp;lt;0.003) and had more often persistent AF (LA, p=0.031) or heart failure (LA, p&amp;lt;0.001). Patients with hypertrophic cardiomyopathy were more often male (LA and RA, p&amp;lt;0.003). Conclusions Fibrotic cardiomyopathy is associated with female sex, persistent AF and heart failure while hypertrophic cardiomyopathy more often occurs in men. More research is needed to establish whether treatment of AF and heart failure prevents the development of atrial cardiomyopathy.

MP-453085-5 ATRIAL FIBRILLATION, FEMALE SEX AND HEART HAILURE ARE THE MAIN DRIVERS OF ATRIAL CARDIOMYOPATHY IN CARDIAC SURGERY PATIENTS: RESULTS FROM THE CATCH ME CONSORTIUM

Atrial cardiomyopathy is emerging as independent prognostic factor in cardiovascular disease. Fibrotic remodeling, cardiomyocyte hypertrophy and a reduction in capillary density are histological hallmarks of atrial cardiomyopathy. The contribution of etiological factors to atrial cardiomyopathy has not been robustly quantified. To quantify the relation between histological features of atrial cardiomyopathy and the clinical profile of patients. We examined left (LAA, n=91) and right (RAA, n=75) atrial appendages sampled from a European cohort of patients undergoing cardiac surgery. Quantification of histological cardiomyopathy features was performed using a recently developed and validated imaging analysis algorithm following myocardial triple staining with wheat germ agglutinin (WGA), CD31 and vimentin. The contributions of AF, heart failure (HF), sex, age, and 4 principal components accounting for confounding clinical characteristics to over-all and endomysial fibrosis were determined in a multivariate model. K-means clustering of 6 atrial histological features was performed to identify different types of remodeling. LAA samples showed less fibrosis (p<0.001), a higher capillary density (p<0.05), a smaller capillary size (p<0.05), and larger cardiomyocytes (p<0.01) than RAA samples. In both LAA and RAA, persistent AF was associated with endomysial fibrosis, while over-all fibrosis was not. Men had larger cardiomyocytes (LAA), while women had a higher degree of endomysial fibrosis (LAA). Heart failure patients had more endomysial and over-all fibrosis (LAA), a higher capillary density (LAA) and capillary size (LAA/RAA). Samples clustered into 2 distinct clusters. One cluster showed fibrotic cardiomyopathy, with more endomysial (p<0.001) and overall fibrosis (p<0.001), more fibroblasts (p<0,001) and a higher capillary density (p<0.001). The other cluster showed hypertrophic cardiomyopathy, with hypertrophic cardiomyocytes (p<0.001). Patients with fibrotic cardiomyopathy were more often female (LAA and RA, p<0.003) and had more often persistent AF (LAA, p=0.031) or heart failure (LAA, p<0.001). Patients with hypertrophic cardiomyopathy were more often male (LAA and RAA, p<0.003). Fibrotic cardiomyopathy is associated with female sex, persistent AF and heart failure while hypertrophic cardiomyopathy more often occurs in men. More research is needed to establish whether treatment of AF and heart failure prevents the development of atrial cardiomyopathy.

Presenilin1 FAD mutants and γ‐secretase inhibitors decrease VEGFR2 signaling and block angiogenic functions of brain endothelial cells

AbstractBackgroundAlzheimer’s disease (AD) is a multifactorial and progressive neurodegenerative disease where cerebrovascular abnormalities are commonly observed. Various factors, including decreased sprouting angiogenesis, can cause a reduction in capillary density observed in AD. In addition, brain vascular alterations occur before the appearance of neuropathology in animal AD models. Brain angiogenesis is a reparative function in response to toxic insults and is regulated by endothelial cells (ECs) and angiogenic factors such as the VEGF; impairment in this function would render the brain vulnerable to insults. In present study, we examine the effects of Presenilin 1 (PS1)‐Familial AD (FAD) mutants and γ‐secretase in the VEGF‐induced angiogenic signaling and functions of brain ECs.MethodPrimary cortical ECs (pCECs) from brains of wild‐type (WT) and knock‐in (KI) mice expressing either PS1 M146V or PS1 I213T FAD mutant in heterozygous state were isolated and cultured. These mice constitute a “humanized” FAD model having the same genotype as human FAD patients. We performed the in vitro angiogenesis assays using pCEC cultures. Processing of VEGFR2 was detected in HEK293 cells overexpressing VEGFR2 in the presence or absence of VEGF‐A and γ‐secretase inhibitor (RO4929097). The same processing was also analyzed by western blotting in PS1 M146V and I213T mutant pCECs treated with VEGF‐A. VEGF‐induced phosphorylation of signaling molecules downstream of VEGFR2 such as p38 kinase and PLCγ1 was also examined in WT and PS1 M146V mutant pCECs by western blotting.ResultWe found that VEGFR2 is processed by γ‐secretase and that this processing together with VEGF‐induced sprouting, tube formation, migration and angiogenic complexes between VE‐cadherin and Rok‐α kinase are decreased in the presence of γ‐secretase inhibitor or by PS1 FAD mutants. Furthermore, the VEGF‐induced phosphorylation of p38 and PLCγ1 is decreased in PS1 FAD mutant pCECs.ConclusionOur findings suggest that VEGFR2 is processed by γ‐secretase and that γ‐secretase promotes VEGF‐induced angiogenic functions of brain ECs through activation of VEGF‐VEGFR2 downstream signaling. Our data also show that a PS1 FAD mutant decreases both the VEGFR2 processing by γ‐secretase and the VEGF‐induced signaling and angiogenic functions of pCECs, providing a mechanism via which FAD mutants affect brain angiogenesis.

Abstract 15180: Cerebral Blood Flow Measured by Mri is a Key Translational Hallmark of Brain Injury in Experimental Models of Hypertension-Induced Cognitive Impairment

While several works pointed out the epidemiological associations between hypertension and dementia, the underlying mechanisms are still relatively unknown. In this work we focused on the application of translational tools like MRI to investigate the brain injury induced by hypertension and characterized by cognitive impairment and neuroinflammation. Cerebral hypertension was induced in mice by transverse aortic constriction (TAC) or sham procedure, as previously demonstrated by our group. We characterized cerebral blood flow (CBF) by 7T MRI, measuring the cerebral tissue blood prefusion. Morris Water Maze (MWM) was used to evaluate cognitive function in hypertensive and normotensive mice. Cortical microvasculature was analyzed in histological brain sections. A group of mice was treated with an antibody neutralizing IFNy, a key proinflammatory cytokine induced by hypertension. TAC mice showed a marked cerebral hypoperfusion, with CBF reduction in cortex (Sham: 96.58 vs TAC: 74.25 mL/100g/min p=0.024) and hypothalamus (Sham: 140 vs TAC: 121.4 mL/100g/min p=0.027). Tissue analysis revealed a reduction in capillary density defined as portion of field of view (FOV) occupied by capillaries (Sham: 11.3% vs TAC: 7.7%, p=0.004) and loss of their integrity in the cortex of hypertensive mice, defined as pericyte coverage percentage in the FOV (Sham: 79% vs TAC: 36%, p=0.01). Finally, the impairment in cognitive function measured by the MWM correlated with the CBF measures (r 2 :0.407, p=0.01). In the end, we subjected TAC mice to anti-IFNy treatment and analyzed the effects on brain vasculature assessed in vivo by MRI and ex vivo by histology. We observed that, while leaving unchanged the hemodynamic challenge on the brain, the treatment induced a global rescue in CBF (TAC: 99.85 vs TAC anti-IFNy: 116 mL/100g/min p=0. 008) and a strong correlation between MRI-measured CBF and microvascular density (r 2 :0.471, p=0.002). In conclusion, CBF evaluated by MRI is a key translational hallmark of hypertensive brain injury, and its evaluation in the clinical setting can provide insights on cerebral microvasculature health.

Myocardial ischaemia in cardiac amyloidosis: a change of perspective

Abstract Introduction Cardiac involvement is the main driver of clinical outcomes in systemic amyloidosis; however many clinical observations are not explained by the concept of replacement of the interstitium by amyloid material. Preliminary studies support the hypothesis that myocardial ischaemia contributes to cellular damage. Purpose This study assesses the presence and mechanisms of myocardial ischaemia using cardiovascular magnetic resonance (CMR) with multiparametric mapping and histopathological assessment. Methods Ninety-two patients with cardiac amyloidosis (CA) (AL = 41, ATTR = 51) and 97 without CA (3-vessel coronary disease (3VD) = 47, unobstructed coronary arteries = 26, healthy volunteers (HV) = 24) underwent quantitative stress perfusion CMR with myocardial blood flow (MBF) mapping. Twenty-six myocardial biopsies and 3 explanted hearts with CA were analysed histopathologically. Results Stress MBF was severely reduced in patients with CA with lower values than patients with 3VD, unobstructed coronary arteries and HV (CA = 1.03±0.51 ml/min/g, 3VD = 1.35±0.50 ml/min/g, Unobstructed coronaries = 2.92±0.52 ml/min/g, HV = 3.14±0.69 ml/min/g; CA vs 3VD p=0.008, CA vs Unobstructed coronaries p&amp;lt;0.001, CA vs HV p&amp;lt;0.001). After adjustment for intracellular volume the MBF in patients with CA remained significantly lower than in HV (stress MBF/ICV: AL = 2.24±1.12, ATTR = 2.22±0.93, HV = 4.38±1.06; AL vs. ATTR p=1.000, AL vs HV p&amp;lt;0.001, ATTR vs. HV p&amp;lt;0.001). Myocardial perfusion reserve (MPR) was severely reduced in CA patients, compared to HV and patients with unobstructed coronary arteries, with the degree of reduction being comparable only to patients with 3VD (CA = 1.55±0.60, 3VD = 1.54±0.51, unobstructed coronaries = 2.78±0.70, HV = 4.08±0.86; CA vs 3VD p=1.000, CA vs unobstructed coronary arteries p&amp;lt;0.001, CA vs. HV p&amp;lt;0.001). Myocardial perfusion abnormalities correlated with amyloid burden, systolic and diastolic function, structural parameters and blood biomarkers (p&amp;lt;0.05). Biopsies demonstrated diffuse hypoxia with abnormal VEGF staining in cardiomyocytes and endothelial cells. Amyloid infiltration in intramural arteries was associated with severe lumen reduction in 20% of vessels, and severe reduction in capillary density. Conclusion CA is associated with severe myocardial ischaemia demonstrable by histology and CMR stress perfusion mapping. Histological evaluation indicates a complex pathophysiology, where systolic and diastolic dysfunction, amyloid infiltration of the epicardial arteries and disruption and rarefaction of the capillaries play a role in contributing to myocardial ischaemia. Funding Acknowledgement Type of funding sources: None.

Skin capillary alterations in patients with acute SarsCoV2 infection.

Acute SarsCov2 infection is associated with endothelial dysfunction and 'endothelitis', which might explain systemic microvascular impairment. The presence of endothelial damage may promote vasoconstriction with organ ischemia, inflammation, tissue oedema and a procoagulant state resulting in an increase in the incidence of cardiovascular and cerebrovascular events. Microvascular thrombosis has been demonstrated in postmortem autopsy of COVID-19 patients; however, few data are available about skin capillary alterations in these patients. We evaluated skin microvascular alteration in 22 patients admitted to our hospital with SarsCov2 infection. Capillary density was evaluated by capillaroscopy in the nailfold and the dorsum of the finger in the acute phase of the disease. Capillaroscopy was repeated after 3 months (recovery phase). In addition, blood chemistry parameters and inflammatory markers were obtained during acute infection and at the recovery after 3 months. Patients with COVID-19 showed skin microvascular complications, such as thrombosis, microhaemorrhages and neoangiogenesis, which were not detected after 3 months from the discharge. A significant reduction of capillary density in the dorsum was observed after 3 months from the acute infection (97.2 ± 5.3 vs. 75.81 ± 3.9 n/mm 2P < 0.05). A significant inverse correlation between C-reactive protein and capillary density was observed in patients with acute SarsCov2 infection ( r = 0.44, P < 0.05). Conversely a direct correlation between capillary density during the acute phase and lymphocyte number was detected ( r = 0.49, P < 0.05). This is the first in-vivo evidence of skin capillary thrombosis, microhaemorrhages and angiogenesis in patients with acute SarsCov2 infection, which disappeared after 3 months, supporting the presence of endothelial dysfunction and inflammation. Capillary alterations might reflect systemic vascular effects of viral infection.

Neurovascular unit pathology is observed very early in disease progression in the mutant SOD1G93A mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disease characterized by motor neuron degeneration that causes neuromuscular denervation, resulting in muscle weakness and atrophy. Work over the decades using ALS mouse models has revealed that while initial pathology may occur within motor neurons, disease pathology is cell non-autologous. Impairment of the blood-spinal cord barrier (BSCB) occurs before motor neuron frank degeneration; however, precisely when the early pathogenesis of the neurovascular units occurs is not fully understood. Here we examine changes in morphology of neurovascular units, associated gene and protein expression in the lumbar spinal cord of SOD1G93A, and wild-type mice and correlate results with previous reports of early pathological events. Using RNA-sequencing and immunolabeling, we also show that both the neurovascular units and the vasculature of the SOD1G93A lumbar spinal cord present important modifications throughout the disease. Genes relevant for the neurovascular unit and immune cells were differentially expressed in the SOD1G93A ventral lumbar spinal cord compared to wild-type. A reduction in capillary density and tight junction (TJ) with overt BSCB breakdown was observed in the SOD1G93A lumbar spinal cord and ultrastructural observation revealed intact TJ. Additionally, thickened basement membrane, increased pericytes, and string vessels were observed. These alterations in neurovascular units and the vasculature are observed prior to reports of initial neuromuscular junction denervation. The identification of early pathogenesis may be critical to develop diagnostic tests and development of novel treatment strategies that target these early events.

Longitudinal optical coherence tomography angiography (OCT-A) in a patient with radiation retinopathy following plaque brachytherapy for uveal melanoma

PurposePatients with choroidal melanoma treated with brachytherapy lose vision over time due to radiation retinopathy and optic neuropathy. Newer imaging modalities such as optical coherence tomography angiography (OCT-A) may provide further insight into the ultrastructural vascular changes that occur over time. We studied the progressive OCT-A derived reduction in capillary density that occurred in the macula and juxtapapillary region of a patient treated with plaque brachytherapy for posterior uveal melanoma. MethodsA patient with medium-sized choroidal melanoma in the inferonasal mid-periphery of the right eye was followed with OCT-A imaging in addition to standard imaging (color fundus photography, standardized echography, OCT) over a four-year time period following brachytherapy. Images were analyzed to measure vascular density in nine discrete areas of the macula at each time point as a function of region-specific radiation dose. ResultsOCT-A over time showed focal capillary loss and enlargement of the foveal avascular zone in addition to vascular re-modeling. These changes progressed over time despite improvement in the clinical markers of radiation retinopathy (cotton wool spots, retinal hemorrhages). Radiation dose significantly correlated with rate of reduction in vascular density assessed within 9 square sectors of the macula, and was greatest in sectors closest to the plaque, which had received the highest radiation dose. There was no change in the choriocapillaris flow area over time. The patient developed cystoid macular edema, but maintained 20/30 vision. Conclusions and ImportanceLongitudinal OCT-A demonstrates the microvascular changes that occur in response to radiation over time. Identification of these features may help define therapeutic windows to prevent vision loss associated with radiation retinopathy and optic neuropathy. Ongoing studies will describe a larger cohort of patients followed with this modality over time.

Habilitation for the Aging Avocational Singer

Habilitation for the Aging Avocational Singer Dann Mitton (bio) VOICE PEDAGOGY We are getting older. Nearly seven million Canadians are currently age 65 or older.1 More than two-fifths of U.S. Baby Boomers have already reached the de facto retirement age of 65; the first Gen Xers to cross this aging milestone will do so in 2030. By 2060, 94.7 million Americans are projected to be age 65 or older.2 Williamson reports that "the fastest growing segment of the population is in the over age 85 category."3 This means a steadily growing market segment for the informed voice teacher who understands and can address the needs of the aging singer. The purpose of this article is to curate some thoughts on voice pedagogy (listening, interventions, and resources) that can equip voice teachers to better support an aging singer clientele. It will identify aspects of the aging process that impact the singing voice, and touch on pedagogic interventions for six common challenges experienced by aging singers: onset, power, intonation, vibrato, noise, and vowel production. An in-depth discussion of serious age-related diseases such as Parkinson's, chronic illness, or vocal pathologies is outside the scope of this article. The assumption is that voice teachers understand and embrace their role as part of a greater voice care team that will include certified medical experts to whom one defers whenever issues of health and singing intersect. WHAT IS AGING? Aging is a complex process in which changes at the molecular, cellular, and organ levels result in a progressive and inevitable decrease in the body's ability to respond appropriately to disruptions.4 [Aging affects] accuracy, speed, endurance, stability, strength, coordination, breathing capacity, nerve conduction velocity, heart output, and kidney function. Muscle and neural tissues atrophy, and the chemicals responsible for nerve transmissions change. Ligaments atrophy, and cartilages turn to bone (including those in the larynx). Joints develop irregularities that interfere with smooth motion. The vocal folds themselves thin and deteriorate, losing their elastic and collagenous fibers. This makes them stiffer and thinner and may correlate with voice changes often noted with aging such as breathiness, slightly decreased volume, "thinning" of the voice, and loss of vocal efficiency … The vocal fold edge also becomes less smooth.5 The loss of muscle mass from aging is also known as sarcopenia. [End Page 347] Johnson and Brunssen correlate these widespread systemic changes to the following effects for the aging singer: loss of range (up and/or down); noise in the sound; breaks in the voice; "knerdly" sound and other compensatory effort; intonation; need to take a few more breaths; vibrato slower; warming up takes longer; unclear open vowels; slower coloratura; less ring and intensity in the sound; vocal fatigue; cramping in voice: ceiling; /h/ between notes; difficulty with soft singing and decrescendos; debilitating and compensatory efforts to make up for compromised function.6 Gerontologist Dr. Tracey Gendron questions the ethics of feeding the larger narrative of aging as a progressive decline.7 "Older age is a time that we can actually look forward to. People really just enjoy themselves more and are at peace with who they are. I would love for everyone to say their age at every year and celebrate it."8 Can one recapture youthful vigor? Exciting new research shows that it soon may be possible to undo the reduction in capillary density that accompanies aging. Capillaries are the vehicles through which oxygen off-loads from the blood to the muscle cells. The greater the density of capillaries, the faster the metabolism is excited.9 A 2018 study on aging mice successfully restored the number of blood capillaries and capillary density seen in younger mice, concomitantly restoring their ability to exercise rigorously as in youth.10 This is encouraging as we develop future therapies that can benefit humans. One manifest advantage enjoyed by the aging performing artist is life experience. Lived experience "allows older singers to bring a level of emotion to a performance that is not possible in youth."11 Another point of optimism is that aspects of the aging process respond very well to intervention. Physical decline does not take place uniformly among the...

Abstract 118: Interleukin 4 And 13 Signaling In Macrophages Regulates Neonatal Cardiac Regeneration

Introduction: Heart failure (HF) is a prevalent disease, projected to affect over 8 million Americans by 2030. Current therapy partially decreases progression; however, mortality and disease burden continue to be high. Therefore, there is an unmet need to develop new strategies that target HF progression. Our lab employs the neonatal mouse model of cardiac regeneration to identify pro-reparative pathways that can be applied to treating HF in humans. Previously we demonstrated that the anti-inflammatory cytokine, Interleukin 13 (IL13), promotes cardiac regeneration, however, the cell types mediating this response remain unknown. IL13 and the related cytokine, IL4, share a common receptor (IL4Rα) and both cytokines polarize macrophages into a reparative phenotype. Here, we hypothesize that IL4/13 signaling to macrophages promotes heart regeneration after cardiac injury and we explore the cell source of these cytokines during neonatal development and cardiac regeneration. Methods and Results: We generated a genetic model whereby IL4Rα is depleted in macrophages by crossing IL4Rα floxed (IL4Rα fl/fl ) mice with transgenic mice CX3Cr1 driven-Cre recombinase (CX3CR1 Cre ). Flow cytometry analysis confirmed depletion of IL4Rα in cardiac macrophages. We performed myocardial infarction (MI) on postnatal day 1 (P1) mice and assessed cardiac regeneration by ultrasonography 21 days post-injury (dpi). We found that IL4Rα fl/fl CX3CR1 Cre mice had lower ejection fraction compared to IL4Rα fl/fl littermate controls. Preliminary results suggest there is a reduced capillary density in peri-ischemic myocardium. In addition, we used fluorescent reporter mouse lines; IL4-enhanced green fluorescent protein (IL4-GFP) and IL13-yellow fluorescent protein (IL13-YFP), to assess the cellular source of IL4 and IL13 expression in the hearts of neonatal mice. In unoperated mice, we found no detectable expression of IL13 by any cell type in the heart, whereas IL4 was expressed in innate lymphoid cells (ILCs) and T cells. 4 days after MI in P1 mice IL13 was upregulated in ILCs and T cells and IL4 was expressed by ILCs and T cells and upregulated in myeloid cells. Conclusions and Discussion: We found that IL13 and IL4 are primarily expressed in ILCs and T cells following neonatal injury, suggesting a novel role for ILCs and T cells in the production of IL4 and 13 during the neonatal regeneration process. In addition, we found that lack of IL4/13 signaling in macrophages via depletion IL4Rα impairs cardiac regeneration after MI in neonatal mice, and results in reduced capillary density in peri-ischemic myocardium. We hypothesize that IL4Rα depletion in macrophages impairs reparative macrophage polarization after MI and promotes an inflammatory polarization. Future studies will be aimed to assess macrophage phenotypes in response to IL13/IL4 signaling by transcriptional profiling and flow cytometry.

Abstract MP27: Endothelial Nogo-B Modulates Ischemia Induced Collateral Artery Remodeling

Nogo-B, a member of the reticulon 4 family of proteins, is the dominant isoform expressed in endothelial cells (EC). We have shown that Nogo-B is necessary for blood flow recovery in ischemia. Mice lacking Nogo-B exhibit reduced arteriogenesis and angiogenesis that are linked to a decrease in macrophage infiltration and inflammatory gene expression in vivo. However, whether endothelial Nogo-B regulates arteriogenesis and/or angiogenesis is unknown. We generated an inducible EC-specific mouse overexpressing Nogo-B (Ng ECtg ) and investigated the arteriogenesis and angiogenesis in limb ischemia. Ischemia increased endothelial and serum Nogo-B expression. Blood flow recovery was markedly diminished after femoral artery ligation (FAL) in Ng ECtg compared to WT mice, in association with lower collateral density accessed by micro-CT arteriography. There was no reduction in capillary density or decrease in smooth muscle/pericyte and macrophage recruitment in Ng ECtg mice, suggesting EC Nogo-B overexpression regulates arteriogenesis but not angiogenesis. While Ng ECtg mice have normal number of native collateral artery, the early remodeling of the collateral artery was impaired after ischemia. Furthermore, in line with impaired remodeling, Ng ECtg mice have decreased functional (exercise) hyperemia response in the non-ischemic limb in vivo, and resistant arteries from Ng ECtg have diminished flow induced vasodilatation ex vivo compared to WT mice. Moreover, endothelial reconstitution of Nogo-B in global Nogo knockout background (NogoKO ECrc ) restored blood flow recovery after ischemia in vivo, further suggesting the EC specific function of Nogo-B in arteriogenesis. Mechanistically, we have shown that EC isolated from Ng ECtg mice have decreased nitric oxide release. Nogo-B overexpression negatively regulates eNOS phosphorylation, expression and golgi localization in EC. Future parabiosis study is warranted to elucidate the contribution of paracrine or EC intrinsic Nogo-B in modulating arteriogenesis and angiogenesis. In conclusion, our data showed for the first time that endothelial Nogo-B plays important roles in regulating collateral artery remodeling and blood flow through, at least in part, regulating NO bioavailability.

Nailfold capillaroscopy changes associated with anti-RNP antibodies in systemic lupus erythematosus.

Anti-U1RNP antibody is associated with distinct organ involvement in patients with systemic lupus erythematosus (SLE). Nailfold capillaroscopy (NFC) allows non-invasive assessment of microvascular abnormalities in several connective tissue diseases. The objective of this study is to determine the association of anti-U1RNP antibody with microvascular changes by NFC in RNP-positive SLE patients in comparison with RNP-negative SLE patients (negative disease controls) and mixed connective tissue disease (MCTD) cases (positive disease controls). NFC examination was performed in consecutive patients with SLE with or without anti-U1RNP positivity. MCTD patients were recruited as disease controls. Abnormalities noted in the three groups were compared using non-parametric tests. Ordinal logistic or linear regression was used wherever applicable. 81 patients were studied, of whom 28 were diagnosed as RNP-positive SLE (age 30.0 ± 10.37; 26 females), 26 were RNP-negative SLE (age 29.42 ± 9.20; 25 females) and 27 had MCTD (age36.5 ± 9.70; 25 females). RNP-positive SLE patients had more frequent giant capillaries, enlarged capillaries and ramified capillaries as compared to RNP-negative SLE (p = 0.05, < 0.01 and 0.03, respectively). The capillary density was lower in patients with MCTD as compared with patients withRNP-positive SLE (5.11 ± 1.69/mm vs 7.25 ± 1.38/ mm, p < 0.01) and RNP-negative SLE (8.92 ± 1.13/mm, p < 0.01). The reduction in capillary density was less severe in patients withRNP-negative SLE as compared with RNP-positive SLE (OR = 0.1058 [95% CI = 0.02-0.546], p < 0.01) which wasindependent of the presence of Raynaud'sphenomenon, interstitial lung disease and disease duration. Presence of anti-U1RNP antibody is associated with notable patterns of microvascular abnormalities in SLE. These NFC abnormalities are noted more profoundly in patients with MCTD and are less marked in RNP-negative SLE patients.

English

BACKGROUND The nailfold capillaroscopy (NFC) has been used to analyse capillary microarchitecture in diseases like Raynaud’s disease, scleroderma, and other collagen disorders earlier but recently researchers have documented capillaroscopic findings in diabetic patients which correspond to their ophthalmoscopic findings. Every diabetic patient after ten years of the disease is at risk of diabetic retinopathy and should thereby be referred to an ophthalmologist for screening. But for various factors the referral to ophthalmologists is poor and the patients usually present to the ophthalmologist when their vision is threatened. Our study intends to correlate the nailfold capillaroscopic microvascular changes with duration of diabetes, severity of diabetic retinopathy (DR), compare the changes of patients with DR and without DR and determine its role as a screening tool. METHODS This was a hospital based cross-sectional study for over 18 months in patients (15 - 75 yrs. of age) with diabetes mellitus. Patients with other vascular disorders were excluded. The study subjects were evaluated for diabetic retinopathy by indirect ophthalmoscopy, fundus pictures and optical coherence tomography. The nailfold capillaroscopy findings were recorded and co-related with fundus findings. Statistical analysis was done by using the STATA software version 15.1. RESULTS Two hundred and fifty patients were recruited with 125 patients in each group, patients with and without DR respectively. Poor glycaemic control was seen most commonly in patients with proliferative diabetic retinopathy. The reduced capillary density, tortuosity, microhaemorrhages, neoangiogenesis and avascular areas were seen more frequently in proliferative diabetic retinopathy than nonproliferative diabetic retinopathy and patients without DR. (P value 0.00). CONCLUSIONS Changes in nailfold capillaroscopy in diabetics have significant association with severity of DR, duration of diabetes, and glycaemic control. It could be used as screening and early diagnostic tool for non-ophthalmology medical fraternities to refer to ophthalmologist for follow-up and treatment of diabetic retinopathy. KEYWORDS Diabetes Mellitus, Diabetic Retinopathy, Nailfold Capillaroscopy, Microangiopathy

Renal tubular dilation and fibrosis after unilateral ureter obstruction revealed by relaxometry and spin-lock exchange MRI.

We evaluated the use of quantitative MRI relaxometry, including the dispersion of spin-lock relaxation with different locking fields, for detecting and assessing tubular dilation and fibrosis in a mouse model of unilateral ureter obstruction (UUO). C57BL/6J and BALB/c mice that exhibit different levels of tubular dilation and renal fibrosis after UUO were subjected to MR imaging at 7T. Mice were imaged before UUO surgery, and at 5, 10 and 15 days after surgery. We acquired maps of relaxation rates and fit the dispersion of spin-lock relaxation rates R1ρ at different locking fields (frequencies) to a model of exchanging water pools, and assessed the sensitivity of the derived quantities for detecting tubular dilation and fibrosis in kidney. Histological scores for tubular dilation and fibrosis, based on luminal space and positive fibrotic areas in sections, were obtained for comparison. Histology detected extensive tubular dilation and mild to moderate fibrosis in the UUO kidneys, in which enlargement of luminal space, deposition of collagen, and reductions in capillary density were observed in the cortex and outer stripe of the outer medulla. Relaxation ratesR1 , R2 and R1ρ clearly decreased in these regions of UUO kidneys longitudinally. While R1 showed the highest detectability to tubular dilation and overall changes in UUO kidneys, Sρ , a parameter derived from R1ρ dispersion data, showed the highest correlation with renal fibrosis in UUO. While relaxation parameters are sensitive to tubular dilation in UUO kidneys, Sρ depends primarily on the average exchange rate between water and other chemically shifted resonances such as hydroxyls and amides, and provides additional specific information for evaluating fibrosis in kidney disease.

Intermittent hypoxia changes the interaction of the kinin-VEGF system and impairs myocardial angiogenesis in the hypertrophic heart.

Intermittent hypoxia (IH) is a feature of obstructive sleep apnea (OSA), a condition highly associated with hypertension‐related cardiovascular diseases. Repeated episodes of IH contribute to imbalance of angiogenic growth factors in the hypertrophic heart, which is key in the progression of cardiovascular complications. In particular, the interaction between vascular endothelial growth factor (VEGF) and the kallikrein‐kinin system (KKS) is essential for promoting angiogenesis. However, researchers have yet to investigate experimental models of IH that reproduce OSA, myocardial angiogenesis, and expression of KKS components. We examined temporal changes in cardiac angiogenesis in a mouse IH model. Adult male C57BI/6 J mice were implanted with Matrigel plugs and subjected to IH for 1–5 weeks with subsequent weekly histological evaluation of vascularization. Expression of VEGF and KKS components was also evaluated. After 3 weeks, in vivo myocardial angiogenesis and capillary density were decreased, accompanied by a late increase of VEGF and its type 2 receptor. Furthermore, IH increased left ventricular myocardium expression of the B2 bradykinin receptor, while reducing mRNA levels of B1 receptor. These results suggest that in IH, an unexpected response of the VEGF and KKS systems could explain the reduced capillary density and impaired angiogenesis in the hypoxic heart, with potential implications in hypertrophic heart malfunction.