Organ Mapping Antibody Panels: a community resource for standardized multiplexed tissue imaging.

Multiplexed antibody-based imaging enables the detailed characterization of molecular and cellular organization in tissues. Advances in the field now allow high-parameter data collection (>60 targets); however, considerable expertise and capital are needed to construct the antibody panels employed by these methods. Organ mapping antibody panels are community-validated resources that save time and money, increase reproducibility, accelerate discovery and support the construction of a Human Reference Atlas.

The role of vasculature and angiogenesis in respiratory diseases.

In European countries, nearly 10% of all hospital admissions are related to respiratory diseases, mainly chronic life-threatening diseases such as COPD, pulmonary hypertension, IPF or lung cancer. The contribution of blood vessels and angiogenesis to lung regeneration, remodeling and disease progression has been increasingly appreciated. The vascular supply of the lung shows the peculiarity of dual perfusion of the pulmonary circulation (vasa publica), which maintains a functional blood-gas barrier, and the bronchial circulation (vasa privata), which reveals a profiled capacity for angiogenesis (namely intussusceptive and sprouting angiogenesis) and alveolar-vascular remodeling by the recruitment of endothelial precursor cells. The aim of this review is to outline the importance of vascular remodeling and angiogenesis in a variety of non-neoplastic and neoplastic acute and chronic respiratory diseases such as lung infection, COPD, lung fibrosis, pulmonary hypertension and lung cancer.

Multidimensional Analysis of the Adult Human Heart in Health and Disease Using Hierarchical Phase-Contrast Tomography.

Background Current clinical imaging modalities such as CT and MRI provide resolution adequate to diagnose cardiovascular diseases but cannot depict detailed structural features in the heart across length scales. Hierarchical phase-contrast tomography (HiP-CT) uses fourth-generation synchrotron sources with improved x-ray brilliance and high energies to provide micron-resolution imaging of intact adult organs with unprecedented detail. Purpose To evaluate the capability of HiP-CT to depict the macro- to microanatomy of structurally normal and abnormal adult human hearts ex vivo. Materials and Methods Between February 2021 and September 2023, two adult human donor hearts were obtained, fixed in formalin, and prepared using a mixture of crushed agar in a 70% ethanol solution. One heart was from a 63-year-old White male without known cardiac disease, and the other was from an 87-year-old White female with a history of multiple known cardiovascular pathologies including ischemic heart disease, hypertension, and atrial fibrillation. Nondestructive ex vivo imaging of these hearts without exogenous contrast agent was performed using HiP-CT at the European Synchrotron Radiation Facility. Results HiP-CT demonstrated the capacity for high-spatial-resolution, multiscale cardiac imaging ex vivo, revealing histologic-level detail of the myocardium, valves, coronary arteries, and cardiac conduction system across length scales. Virtual sectioning of the cardiac conduction system provided information on fatty infiltration, vascular supply, and pathways between the cardiac nodes and adjacent structures. HiP-CT achieved resolutions ranging from gross (isotropic voxels of approximately 20 µm) to microscopic (approximately 6.4-µm voxel size) to cellular (approximately 2.3-µm voxel size) in scale. The potential for quantitative assessment of features in health and disease was demonstrated. Conclusion HiP-CT provided high-spatial-resolution, three-dimensional images of structurally normal and diseased ex vivo adult human hearts. Whole-heart image volumes were obtained with isotropic voxels of approximately 20 µm, and local regions of interest were obtained with resolution down to 2.3-6.4 µm without the need for sectioning, destructive techniques, or exogenous contrast agents. Published under a CC BY 4.0 license Supplemental material is available for this article. See also the editorial by Bluemke and Pourmorteza in this issue.

Inflammation and vascular remodeling in COVID-19 hearts.

A wide range of cardiac symptoms have been observed in COVID-19 patients, often significantly influencing the clinical outcome. While the pathophysiology of pulmonary COVID-19 manifestation has been substantially unraveled, the underlying pathomechanisms of cardiac involvement in COVID-19 are largely unknown. In this multicentre study, we performed a comprehensive analysis of heart samples from 24 autopsies with confirmed SARS-CoV-2 infection and compared them to samples of age-matched Influenza H1N1 A (n = 16), lymphocytic non-influenza myocarditis cases (n = 8), and non-inflamed heart tissue (n = 9). We employed conventional histopathology, multiplexed immunohistochemistry (MPX), microvascular corrosion casting, scanning electron microscopy, X-ray phase-contrast tomography using synchrotron radiation, and direct multiplexed measurements of gene expression, to assess morphological and molecular changes holistically. Based on histopathology, none of the COVID-19 samples fulfilled the established diagnostic criteria of viral myocarditis. However, quantification via MPX showed a significant increase in perivascular CD11b/TIE2 + -macrophages in COVID-19 over time, which was not observed in influenza or non-SARS-CoV-2 viral myocarditis patients. Ultrastructurally, a significant increase in intussusceptive angiogenesis as well as multifocal thrombi, inapparent in conventional morphological analysis, could be demonstrated. In line with this, on a molecular level, COVID-19 hearts displayed a distinct expression pattern of genes primarily coding for factors involved in angiogenesis and epithelial-mesenchymal transition (EMT), changes not seen in any of the other patient groups. We conclude that cardiac involvement in COVID-19 is an angiocentric macrophage-driven inflammatory process, distinct from classical anti-viral inflammatory responses, and substantially underappreciated by conventional histopathologic analysis. For the first time, we have observed intussusceptive angiogenesis in cardiac tissue, which we previously identified as the linchpin of vascular remodeling in COVID-19 pneumonia, as a pathognomic sign in affected hearts. Moreover, we identified CD11b + /TIE2 + macrophages as the drivers of intussusceptive angiogenesis and set forward a putative model for the molecular regulation of vascular alterations.

Pulmonary Vascular Endothelialitis, Thrombosis, and Angiogenesis in Covid-19.

BACKGROUND: Progressive respiratory failure is the primary cause of death in the coronavirus disease 2019 (Covid-19) pandemic. Despite widespread interest in the pathophysiology of the disease, relatively little is known about the associated morphologic and molecular changes in the peripheral lung of patients who die from Covid-19. METHODS: We examined 7 lungs obtained during autopsy from patients who died from Covid-19 and compared them with 7 lungs obtained during autopsy from patients who died from acute respiratory distress syndrome (ARDS) secondary to influenza A(H1N1) infection and 10 age-matched, uninfected control lungs. The lungs were studied with the use of seven-color immunohistochemical analysis, micro-computed tomographic imaging, scanning electron microscopy, corrosion casting, and direct multiplexed measurement of gene expression. RESULTS: In patients who died from Covid-19-associated or influenza-associated respiratory failure, the histologic pattern in the peripheral lung was diffuse alveolar damage with perivascular T-cell infiltration. The lungs from patients with Covid-19 also showed distinctive vascular features, consisting of severe endothelial injury associated with the presence of intracellular virus and disrupted cell membranes. Histologic analysis of pulmonary vessels in patients with Covid-19 showed widespread thrombosis with microangiopathy. Alveolar capillary microthrombi were 9 times as prevalent in patients with Covid-19 as in patients with influenza (P<0.001). In lungs from patients with Covid-19, the amount of new vessel growth - predominantly through a mechanism of intussusceptive angiogenesis - was 2.7 times as high as that in the lungs from patients with influenza (P<0.001). CONCLUSIONS: In our small series, vascular angiogenesis distinguished the pulmonary pathobiology of Covid-19 from that of equally severe influenza virus infection. The universality and clinical implications of our observations require further research to define. (Funded by the National Institutes of Health and others.).

The Challenge of Long-Term Cultivation of Human Precision-Cut Lung Slices.

Human precision-cut lung slices (PCLS) have proven to be an invaluable tool for numerous toxicologic, pharmacologic, and immunologic studies. Although a cultivation period of <1 week is sufficient for most studies, modeling of complex disease mechanisms and investigating effects of long-term exposure to certain substances require cultivation periods that are much longer. So far, data regarding tissue integrity of long-term cultivated PCLS are incomplete. More than 1500 human PCLS from 16 different donors were cultivated under standardized, serum-free conditions for up to 28 days and the viability, tissue integrity, and the transcriptome was assessed in great detail. Even though viability of PCLS was well preserved during long-term cultivation, a continuous loss of cells was observed. Although the bronchial epithelium was well preserved throughout cultivation, the alveolar integrity was preserved for about 2 weeks, and the vasculatory system experienced significant loss of integrity within the first week. Furthermore, ciliary beat in the small airways gradually decreased after 1 week. Interestingly, keratinizing squamous metaplasia of the alveolar epithelium with significantly increasing manifestation were found over time. Transcriptome analysis revealed a significantly increased immune response and significantly decreased metabolic activity within the first 24 hours after PCLS generation. Overall, this study provides a comprehensive overview of histomorphologic and pathologic changes during long-term cultivation of PCLS.

A Morphomolecular Approach to Alveolar Capillary Dysplasia.

Alveolar capillary dysplasia (ACD) is a rare lung developmental disorder leading to persistent pulmonary arterial hypertension and fatal outcomes in newborns. The current study analyzed the microvascular morphology and the underlying molecular background of ACD. One ACD group (n = 7), one pulmonary arterial hypertension group (n = 20), and one healthy con1trol group (n = 16) were generated. Samples of histologically confirmed ACD were examined by exome sequencing and array-based comparative genomic hybridization. Vascular morphology was analyzed using scanning electron microscopy of microvascular corrosion casts. Gene expression and biological pathways were analyzed using two panels on inflammation/kinase-specific genes and a comparison analysis tool. Compartment-specific protein expression was analyzed using immunostaining. In ACD, there was an altered capillary network, a high prevalence of intussusceptive angiogenesis, and increased activity of C-X-C motif chemokine receptor 4 (CXCR4), hypoxia-inducible factor 1α (HIF1A), and angiopoietin signaling pathways compared with pulmonary arterial hypertension/healthy controls. Histologically, there was a markedly increased prevalence of endothelial tyrosine kinase receptor (TEK/TIE2)+ macrophages in ACD, compared with the other groups, whereas the CXCR4 ligand CXCL12 and HIF1A showed high expression in all groups. ACD is characterized by dysfunctional capillaries and a high prevalence of intussusceptive angiogenesis. The results indicate that endothelial CXCR4, HIF1A, and angiopoietin signaling as well as TIE2+ macrophages are crucial for the induction of intussusceptive angiogenesis and vascular remodeling. Future studies should address the use of anti-angiogenic agents in ACD, where TIE2 appears as a promising target.

Vitamin A preserves cardiac energetic gene expression in a murine model of diet-induced obesity.

Perturbed vitamin-A metabolism is associated with type 2 diabetes and mitochondrial dysfunction that are pathophysiologically linked to the development of diabetic cardiomyopathy (DCM). However, the mechanism, by which vitamin A might regulate mitochondrial energetics in DCM has previously not been explored. To test the hypothesis that vitamin-A deficiency accelerates the onset of cardiomyopathy in diet-induced obesity (DIO), we subjected mice with lecithin retinol acyltransferase (Lrat) germline deletion, which exhibit impaired vitamin-A stores, to vitamin A-deficient high-fat diet (HFD) feeding. Wild-type mice fed with a vitamin A-sufficient HFD served as controls. Cardiac structure, contractile function, and mitochondrial respiratory capacity were preserved despite vitamin-A deficiency following 20 wk of HFD feeding. Gene profiling by RNA sequencing revealed that vitamin A is required for the expression of genes involved in cardiac fatty acid oxidation, glycolysis, tricarboxylic acid cycle, and mitochondrial oxidative phosphorylation in DIO as expression of these genes was relatively preserved under vitamin A-sufficient HFD conditions. Together, these data identify a transcriptional program, by which vitamin A preserves cardiac energetic gene expression in DIO that might attenuate subsequent onset of mitochondrial and contractile dysfunction.NEW & NOTEWORTHY The relationship between vitamin-A status and the pathogenesis of diabetic cardiomyopathy has not been studied in detail. We assessed cardiac mitochondrial respiratory capacity, contractile function, and gene expression by RNA sequencing in a murine model of combined vitamin-A deficiency and diet-induced obesity. Our study identifies a role for vitamin A in preserving cardiac energetic gene expression that might attenuate subsequent development of mitochondrial and contractile dysfunction in diet-induced obesity.

Correction of heat-induced susceptibility changes in respiratory-triggered 2D-PRF-based thermometry for monitoring of magnetic resonance-guided hepatic microwave ablation in a human-like in vivo porcine model.

PURPOSE: To develop and evaluate susceptibility corrected 2D proton resonance frequency (PRF)-based magnetic resonance (MR)-thermometry for the accurate assessment of the ablation zone of hepatic microwave ablation (MWA). METHODS AND MATERIALS: Twelve hepatic MWA were performed in five LEWE minipigs with human-like fissure-free liver. Temperature maps during ablation of PRF-based MR-thermometry were corrected by modeling heat induced susceptibility changes. Ablation zones were determined using cumulative equivalent minutes at 43 °C (CEM43) as tissue damage model. T1 weighted (w) post-ablation contrast-enhanced (CE) MR-imaging and manually segmented postmortem histology were used for validation. The agreement of uncorrected (raw) and susceptibility corrected (corr) MR-thermometry with T1w post-ablation CE MR-imaging and histology was evaluated. The Wilcoxon-signed rank test and Bland-Altman analysis were applied. RESULTS: With the susceptibility corrected MR-thermometry a significantly increased dice coefficient (raw: 77% vs. corr: 83%, p < 0.01) and sensitivity (raw: 72% vs. corr: 82%, p < 0.01) was found for the comparison to T1w-CE imaging as well as histopathology (dice coefficients: raw: 76% vs. corr: 79%, p < 0.001; sensitivity: raw: 72% vs. corr: 74%, p < 0.001). While major axis length was significantly increased (7.1 mm, p < 0.001) and minor axis length significantly decreased (2.2 mm, p < 0.001) in uncorrected MR-thermometry compared to T1w-CE MR-imaging, no significant bias was found after susceptibility correction. CONCLUSION: Using susceptibility corrected 2D PRF-based MR-thermometry to predict the ablation zones of hepatic MWA provided a good agreement in comparison to T1w post-ablation CE MR-imaging and histopathology.

Time-Dependent Molecular Motifs of Pulmonary Fibrogenesis in COVID-19.

(1) Background: In COVID-19 survivors there is an increased prevalence of pulmonary fibrosis of which the underlying molecular mechanisms are poorly understood; (2) Methods: In this multicentric study, n = 12 patients who succumbed to COVID-19 due to progressive respiratory failure were assigned to an early and late group (death within ≤7 and >7 days of hospitalization, respectively) and compared to n = 11 healthy controls; mRNA and protein expression as well as biological pathway analysis were performed to gain insights into the evolution of pulmonary fibrogenesis in COVID-19; (3) Results: Median duration of hospitalization until death was 3 (IQR25-75, 3-3.75) and 14 (12.5-14) days in the early and late group, respectively. Fifty-eight out of 770 analyzed genes showed a significantly altered expression signature in COVID-19 compared to controls in a time-dependent manner. The entire study group showed an increased expression of BST2 and IL1R1, independent of hospitalization time. In the early group there was increased activity of inflammation-related genes and pathways, while fibrosis-related genes (particularly PDGFRB) and pathways dominated in the late group; (4) Conclusions: After the first week of hospitalization, there is a shift from pro-inflammatory to fibrogenic activity in severe COVID-19. IL1R1 and PDGFRB may serve as potential therapeutic targets in future studies.

[Interstitial lung disease in infancy and early childhood]. / Nichtneoplastische Lungenerkrankungen des Säuglings und Kindesalters.

Diffuse interstitial lung disease of infancy (chILD) shows a spectrum of disease substantially different from that of adults. Established classification systems divide chILD into conditions that are more prevalent in infancy and conditions that occur at any age. The classification is based on a multidisciplinary approach including clinical, radiological, genetic, and histological findings. Lung biopsies become necessary if other diagnostic investigations have not identified a precise chILD or if severe or refractory respiratory distress of unknown cause is present. As the majority of pediatric lung biopsies will be received first by pathologists outside of specialist centers this review summarizes relevant clinical and histological findings of chILD.

[Fibrotic remodeling of the lung following lung and stem-cell transplantation]. / Fibrotischer Lungenparenchymumbau nach Lungen- und Stammzelltransplantation.

Transplantation of solid organs and hematopoietic stem cells represents an important therapeutic option for a variety of end-stage pulmonary diseases, aggressive hematopoietic neoplasms, or severe immunodeficiencies. Although the overall survival following transplantation has generally improved over recent decades, long-time survival of lung and stem-cell transplant recipients is still alarmingly low with an average 5­year survival rate of only 50-60%. Chronic allo-immunoreactions in general and pulmonary allo-immunoreactions with subsequent fibrosis in particular are major reasons for this poor outcome. Comparable patterns of fibrotic lung remodeling are observed following both lung and hematopoietic stem-cell transplantation. Besides the meanwhile well-established obliterative and functionally obstructive remodeling of the small airways - obliterative bronchiolitis - a specific restrictive subform of fibrosis, namely alveolar fibroelastosis, has been identified. Despite their crucial impact on patient outcome, both entities can be very challenging to detect by conventional histopathological analysis. Their underlying mechanisms are considered overreaching aberrant repair attempts to acute lung injuries with overactivation of (myo-) fibroblasts and excessive and irreversible deposition of extracellular matrix. Of note, the underlying molecular mechanisms are widely divergent between these two morphological entities and are independent of the underlying clinical setting.Further comprehensive investigations of these fibrotic alterations are key to the development of much-needed predictive diagnostics and curative concepts, considering the high mortality of pulmonary fibrosis following transplantation.

[COVID-19: effects on the lungs and heart]. / COVID-19: Auswirkungen auf Lunge und Herz.

Viral respiratory diseases constitute the most common reasons for hospitalization with more than half of all acute illnesses worldwide. Progressive respiratory failure with pronounced diffuse alveolar damage has been identified as the primary cause of death in COVID-19. COVID-19 pneumonia shares common histopathological hallmarks with influenza (H1N1)-related ARDS, like diffuse alveolar damage (DAD) with edema, hemorrhage, and intra-alveolar fibrin deposition. The lungs with COVID-19 pneumonia revealed perivascular inflammation, an endothelial injury, microangiopathy, and an aberrant blood vessel neoformation by intussusceptive angiogenesis. While this pronounced angiocentric inflammation is likely be found - to varying degrees - in numerous other organs, e.g., the heart, COVID-19 is hypothesized to be not just a pulmonary, but rather a systemic "vascular disease."

Dual Function of iPSC-Derived Pericyte-Like Cells in Vascularization and Fibrosis-Related Cardiac Tissue Remodeling In Vitro.

Myocardial interstitial fibrosis (MIF) is characterized by excessive extracellular matrix (ECM) deposition, increased myocardial stiffness, functional weakening, and compensatory cardiomyocyte (CM) hypertrophy. Fibroblasts (Fbs) are considered the principal source of ECM, but the contribution of perivascular cells, including pericytes (PCs), has gained attention, since MIF develops primarily around small vessels. The pathogenesis of MIF is difficult to study in humans because of the pleiotropy of mutually influencing pathomechanisms, unpredictable side effects, and the lack of available patient samples. Human pluripotent stem cells (hPSCs) offer the unique opportunity for the de novo formation of bioartificial cardiac tissue (BCT) using a variety of different cardiovascular cell types to model aspects of MIF pathogenesis in vitro. Here, we have optimized a protocol for the derivation of hPSC-derived PC-like cells (iPSC-PCs) and present a BCT in vitro model of MIF that shows their central influence on interstitial collagen deposition and myocardial tissue stiffening. This model was used to study the interplay of different cell types-i.e., hPSC-derived CMs, endothelial cells (ECs), and iPSC-PCs or primary Fbs, respectively. While iPSC-PCs improved the sarcomere structure and supported vascularization in a PC-like fashion, the functional and histological parameters of BCTs revealed EC- and PC-mediated effects on fibrosis-related cardiac tissue remodeling.

Immunohistochemical marker profiles for the differentiation of collagenous spherulosis from adenoid cystic carcinoma of the breast.

Collagenous spherulosis (CS) is a rare breast lesion of unknown histogenesis. Adenoid cystic carcinoma (ACC) is a rare basal-like breast carcinoma with low histological grade. CS is a benign lesion but resembles ACC. Both lesions show a similar histomorphology and feature bilineage differentiation. This study compared immunohistochemical markers in CS and ACC. We compiled n = 13 CS cases and n = 18 mammary ACCs. Fourteen marker proteins (ER, PR, HER2, GATA3, CK7, E-cadherin, CD117, CK5/14, p40, p63, SMA, CD10, calponin, P-cadherin) were evaluated by immunohistochemistry (IHC). MYB rearrangement, a common alteration in ACC, was assessed by fluorescence in situ hybridization. Patient age ranged between 40-60 years for CS lesions and 30-90 years for ACCs. 7/13 (54%) CS cases harbored a lobular carcinoma in situ (LCIS) in the luminal component. One CS/LCIS lesion occurred in a carrier of a pathogenic germline variant in CDH1/E-cadherin. MYB rearrangement was detected in 0/11 (0%) CS and 6/16 (37%) ACC cases (P = 0.054). CS was associated with expression of ER in the luminal component (P < 0.001), E-cadherin loss in the luminal component (P = 0.045), and expression of CD10 and calponin in the basal component (P < 0.001). Furthermore, CS was associated with GATA3 expression in the luminal component (12/13 [92%] versus 5/18 [27%], P < 0.001). In summary, IHC for GATA3 and E-cadherin may contribute to the differential diagnosis between CS and ACC, although these markers are not exclusively expressed in either lesion. Histologic evaluation has to take into account that CS is frequently colonized by LCIS, requiring thorough correlation of histomorphology and immunohistochemical features.

Hypoxia Attenuates Pressure Overload-Induced Heart Failure.

BACKGROUND: Alveolar hypoxia is protective in the context of cardiovascular and ischemic heart disease; however, the underlying mechanisms are incompletely understood. The present study sought to test the hypothesis that hypoxia is cardioprotective in left ventricular pressure overload (LVPO)-induced heart failure. We furthermore aimed to test that overlapping mechanisms promote cardiac recovery in heart failure patients following left ventricular assist device-mediated mechanical unloading and circulatory support. METHODS AND RESULTS: We established a novel murine model of combined chronic alveolar hypoxia and LVPO following transverse aortic constriction (HxTAC). The HxTAC model is resistant to cardiac hypertrophy and the development of heart failure. The cardioprotective mechanisms identified in our HxTAC model include increased activation of HIF (hypoxia-inducible factor)-1α-mediated angiogenesis, attenuated induction of genes associated with pathological remodeling, and preserved metabolic gene expression as identified by RNA sequencing. Furthermore, LVPO decreased Tbx5 and increased Hsd11b1 mRNA expression under normoxic conditions, which was attenuated under hypoxic conditions and may induce additional hypoxia-mediated cardioprotective effects. Analysis of samples from patients with advanced heart failure that demonstrated left ventricular assist device-mediated myocardial recovery revealed a similar expression pattern for TBX5 and HSD11B1 as observed in HxTAC hearts. CONCLUSIONS: Hypoxia attenuates LVPO-induced heart failure. Cardioprotective pathways identified in the HxTAC model might also contribute to cardiac recovery following left ventricular assist device support. These data highlight the potential of our novel HxTAC model to identify hypoxia-mediated cardioprotective mechanisms and therapeutic targets that attenuate LVPO-induced heart failure and mediate cardiac recovery following mechanical circulatory support.