Biology subjects

Grandoch, M.

Publications and source records attributed to Grandoch, M..

6 recordsLinked to original sources

bioRxiv2024

Novel fluorescence-based methods to determine infarct and scar size in murine models of reperfused myocardial infarction

BackgroundDetermination of infarct area and scar size following myocardial infarction (MI) is commonly used to evaluate the efficacy of potential cardioprotective treatments in mice and other animal models. MethodsFor both, early and late time points following MI, we compared classical histochemical approaches with fluorescence staining methods. Reperfused MI was induced in male C57BL/6J mice and hearts were extracted 24 hours, 7-, 21-, or 28-days following MI and stained with 2,3,5-Triphenyltetrazolium chloride (TTC) and Evans Blue, Hoechst, phalloidin, Sirius Red, Massons or Gomoris Trichrome or Wheat Germ Agglutinin (WGA). ResultsFluorescent staining combining Hoechst and phalloidin constitutes an alternative for TTC and Evans Blue, enabling a clear visualization of infarct area, area at risk, as well as remote area unaffected by MI. Infarct size early after reperfusion determined with TTC staining correlates strongly with that demarcated by phalloidin while combination of Hoechst and phalloidin staining can emulate classical TTC/Evans Blue staining 24 h post-MI. Moreover, WGA is equally accurate as the classical Sirius Red, Massons and Gomoris Trichrome stainings in identifying scar size in later phases (>7d) post-MI. Finally, we demonstrate feasibility of combining conventional fluorescence staining by localizing CD45+ leukocytes to specific regions of the infarcted myocardium. ConclusionWe established staining procedure is not inferior to classical TTC staining while providing substantial benefits including the option for unbiased software-assisted analysis while sparing ample residual tissue for additional analyses. Overall, this enhances the data quality and reduces the required animal numbers consistent with the 3R concept of animal experimentation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/604384v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@17ccc83org.highwire.dtl.DTLVardef@56d433org.highwire.dtl.DTLVardef@857f37org.highwire.dtl.DTLVardef@63297d_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗

bioRxiv2023

Collagen receptor GPVI-mediated platelet activation and pro-coagulant activity aggravates inflammation and aortic wall remodelling in abdominal aortic aneurysm

Platelets play an important role in cardio- and cerebrovascular diseases. Abdominal aortic aneurysm (AAA) is a highly lethal, atherosclerotic-related disease with characteristic features of progressive dilatation of the abdominal aorta and degradation of the vessel wall accompanied by chronic inflammation. Platelet activation and pro-coagulant activity play a decisive role in the AAA pathology as they might trigger AAA development in both mice and men. The present study investigated the impact of the major platelet collagen receptor glycoprotein (GP)VI in cellular processes underlying AAA initiation and progression. Genetic deletion of GPVI offered protection of mice against aortic diameter expansion in experimental AAA. Mechanistically, GPVI deficiency resulted in decreased inflammation with reduced infiltration of neutrophils and platelets into the aortic wall. Further, remodelling of the aortic wall was improved in absence of GPVI, indicated by reduced MMP2/9 and OPN plasma levels and an enhanced -SMA content within the aortic wall, accompanied by reduced cell apoptosis. As a result, an elevation in intima/media thickness and elastin content were observed in GPVI-deficient PPE mice, coursing a significantly reduced aortic diameter expansion and reduced aneurysm incidence. In AAA patients, enhanced plasma levels of soluble GPVI and fibrin, besides fibrin accumulation within the intraluminal thrombus (ILT) suggested that GPVI might serve as a biomarker and mediator in fibrin-supported stabilization of the ILT. In conclusion, our results emphasize the potential need for a GPVI-targeted anti-platelet therapy to reduce AAA initiation and progression, as well as to protect AAA patients from aortic rupture. Translational perspectiveAbdominal aortic aneurysm (AAA) is an atherosclerotic-related, cardiovascular disease (CVD) with high mortality. The impact of platelets in different cellular processes underlying AAA initiation and progression remains unclear.Therefore, we analysed the role of the major platelet collagen receptor GPVI in the pathogenesis of AAA. Results from platelet depleted mice and patients with AAA revealed a significant contribution of GPVI to the inflammatory response and remodelling process of the aorta. Further, elevated accumulation of fibrin, a recently identified ligand of GPVI in the intraluminal thrombus (ILT) and in the plasma of AAA patients, suggests that GPVI binding to fibrin plays a role in ILT formation and probably stabilization of the abdominal aorta. Furthermore, increased levels of sGPVI suggest that GPVI might serve as a clinical biomarker for AAA. Thus, therapeutic targeting of GPVI-mediated platelet activation might be an effective anti-thrombotic strategy for AAA patients.

cell biology↗

bioRxiv2023

Inhibition of hyaluronan synthesis prevents β-cell loss in obesity-associated type 2 diabetes

Pancreatic {beta}-cell dysfunction and death are central to the pathogenesis of type 2 diabetes (T2D). We have identified a novel role for the inflammatory extracellular matrix polymer hyaluronan (HA) in this pathophysiology. Low levels of HA are present in healthy pancreatic islets. However, HA substantially accumulates in cadaveric islets of human T2D and islets of the db/db mouse model of T2D in response to hyperglycemia. Treatment with 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis, or the deletion of the major HA receptor CD44, preserve glycemic control and insulin levels in db/db mice despite ongoing weight gain, indicating a critical role for this pathway in T2D pathogenesis. 4-MU treatment and the deletion of CD44 likewise preserve glycemic control in other settings of {beta}-cell injury including streptozotocin treatment and islet transplantation. Mechanistically, we find that 4-MU increases the expression of the apoptosis inhibitor survivin, a downstream transcriptional target of CD44 dependent on HA/CD44 signaling, on {beta}-cells such that caspase 3 activation does not result in {beta}-cell apoptosis. These data indicate a role for HA accumulation in diabetes pathogenesis and suggest that it may be a viable target to ameliorate {beta}-cell loss in T2D. These data are particularly exciting, because 4-MU is already an approved drug (also known as hymecromone), which could accelerate translation of these findings to clinical studies.

immunology↗

bioRxiv2022

Deficiency in hyaluronan synthase 3 attenuates ruptures in a murine model of abdominal aortic aneurysms by reduced aortic monocyte infiltration

Abdominal aortic aneurysms (AAA) are a common vascular disorder with a high mortality due to the prevalence of aortic ruptures. The underlying pathomechanisms are complex and involve immune cell infiltration and degradation of the vascular extracellular matrix (ECM). Hyaluronan (HA), synthesized at the plasma membrane by three HA synthase isoenzymes (HAS1-3), is not only a major constituent of the ECM but also known to directly affect the phenotype of vascular smooth muscle cells as well as immunological responses. Specifically, the HAS3 isoenzyme has been reported to play a major role in various inflammatory conditions. Therefore, the aim of the present study was to elucidate the role of HAS3-derived HA in the pathogenesis of abdominal aortic aneurysm. To this end, we used a murine model of Angiotensin II (AngII)-induced abdominal aortic aneurysms and dissections (AAAs/AADs) and could demonstrate that genetic depletion of Has3 improves survival in Apoe/Has3 double deficient (Apoe/Has3-DKO) mice via the reduced occurrence of aortic ruptures. Mechanistically, fewer elastica breaks were observed in Apoe/Has3-DKO mice compared to Apoe-KO littermates. This was associated with a decreased infiltration of myeloid immune cells into the vessel wall of Has3-deficient mice while in parallel elevated numbers of circulating leukocytes were detected. RNA seq analysis from aortic tissue pointed towards a disturbed endothelial-myeloid cell communication as a cause for the diminished recruitment of immune cells to the aortic wall. While endothelial cells were unaffected, upregulation of adhesion receptors as well as the HA receptor CD44, known to mediate leukocyte adhesion to the endothelium, was blunted in monocytes from Apoe/Has3-DKO mice in response to AngII treatment. These findings underline the pivotal detrimental role of monocytes HAS3-dependent pericellular HA matrix for an exaggerated immune cell recruitment to inflammatory foci giving here rise for an increased incidence of ruptured aortic aneurysms.

immunology↗

bioRxiv2022

Targeting of MMP-13 prevents aortic aneurysm formation in Marfan mice

Fibrillin-1 assembles into microfibrils that not only define the structural integrity and biomechanics of the aorta but also target and sequester growth factors within the extracellular microenvironment of aortic resident cells. To better understand how dominant negative effects on fibrillin microfibril stability manifest in growth factor driven aortic disease, we analyzed early events of aortic aneurysm formation within the first two weeks of postnatal life in the dominant negative Fbn1 GT8 Marfan mouse model. Echocardiography analysis of homozygous GT8 Fbn1 mice showed significant aortic root enlargement within the second week of postnatal life which correlated with the onset of fibrillin-1 fiber degradation, aberrantly increased BMP activity and upregulated transcript levels of the collagenase MMP-13. We also found the aortic collagen network structurally disturbed where the mutant GT8-fibrillin-1 was detected. Genetic ablation or pharmacological inhibition of MMP-13 in Fbn1 GT8 Marfan mice prevents aortic root dilatation implicating the relevance of this mechanism in aortic aneurysm formation in Marfan syndrome.

biochemistry↗

bioRxiv2021

Single-cell transcriptomics defines heterogeneity of epicardial cells and fibroblasts within the infarcted heart

In the adult heart, the epicardium becomes activated after injury, contributing to cardiac healing by secretion of paracrine factors. Here we analyzed by single-cell RNA sequencing combined with RNA in situ hybridization and lineage tracing of WT1+ cells the cellular composition, location, and hierarchy of epicardial stromal cells (EpiSC) in comparison to activated myocardial fibroblasts/stromal cells in infarcted mouse hearts. We identified 11 transcriptionally distinct EpiSC populations, that can be classified in three groups each containing a cluster of proliferating cells. Two groups expressed cardiac specification makers and sarcomeric proteins suggestive of cardiomyogenic potential. Transcripts of HIF-1 and HIF-responsive genes were enriched in EpiSC consistent with an epicardial hypoxic niche. Expression of paracrine factors was not limited to WT1+ cells but was a general feature of activated cardiac stromal cells. Our findings provide the cellular framework by which myocardial ischemia may trigger in EpiSC the formation of cardioprotective/regenerative responses.

cell biology↗