Biology subjects

Hirschi, K. K.

Publications and source records attributed to Hirschi, K. K..

10 recordsLinked to original sources

bioRxiv2025

Zonal endothelial cell heterogeneity underlies murine renal vascular development.

The renal vasculature consists of highly specialized blood vessels that carry out distinct physiological functions. Defining their transcriptional signatures and tracing their developmental ontogeny has thus far been challenging due to a lack of regionally specific endothelial biomarkers. Here, we performed single nuclear RNA sequencing (snucRNA-Seq) to interrogate embryonic renal endothelial cells. We identified nine different endothelial cell types, and validated expression of novel candidates using multiplex RNAScope. We interrogate biological characteristics, including cell cycle stage, of developmental blood vessels with a variety of in vivo tools. Using genetic tools based on expression of tip cell marker Esm1, we performed detailed lineage tracing, demonstrating multi-clonal and multi-directional endothelial contribution to glomerular vasculature. Together, this study provides the first validated, tool-focused developmental atlas of the murine renal vasculature and elucidates several novel cellular mechanisms of nephron vascularization.

developmental biology↗

bioRxiv2023

Loss of endothelial cell heterogeneity in arteries after obesogenic diet

BackgroundIt is well recognized that obesity leads to arterial endothelial dysfunction and cardiovascular disease. However, the progression to endothelial dysfunction is not clear. Endothelial cells (ECs) adapt to the unique needs of their resident tissue and respond to systemic metabolic perturbations. We sought to better understand how obesity affects EC phenotypes in different tissues specifically focusing on mitochondrial gene expression. MethodsWe performed bulk RNA sequencing (RNA-seq) and single cell RNA-seq (scRNA-seq) on mesenteric and adipose ECs isolated from normal chow (NC) and high fat diet (HFD) fed mice. Differential gene expression, gene ontology pathway, and transcription factor analyses were performed. We further investigated our hypothesis in humans using published human adipose single nuclei RNA-seq (snRNA-seq) data. ResultsBulk RNA-seq revealed higher mitochondrial gene expression in adipose ECs compared to mesenteric ECs in both NC and HFD mice. We then performed scRNA-seq and categorized EC clusters as arterial, capillary, venous, or lymphatic. HFD decreased the number of differentially expressed genes between mesenteric and adipose ECs in all subtypes, but the largest effect was seen in arterial ECs. Further analysis of arterial ECs revealed genes coding for mitochondrial oxidative phosphorylation proteins were enriched in adipose compared to mesentery under NC conditions. In HFD mice, these genes were decreased in adipose ECs becoming similar to mesenteric ECs. Transcription factor analysis revealed C/EBP and PPAR{gamma}, both known to regulate lipid handling and metabolism, had high specificity scores in the NC adipose artery ECs. These findings were recapitulated in snRNA-seq data from human adipose. ConclusionsThese data suggest mesenteric and adipose arterial ECs metabolize lipids differently and the transcriptional phenotype of these two vascular beds converge in obesity, in part, due to downregulation of PPAR{gamma} and C/EBP in adipose artery ECs. This work lays the foundation for investigating vascular bed specific adaptations to obesity.

molecular biology↗

bioRxiv2022

Mechanisms of vascular maturation and maintenance captured by longitudinal imaging of live mouse skin

A functional network of blood vessels is essential for organ growth and homeostasis. Yet, how the vasculature matures and maintains adult homeostasis remains elusive in live mice. By longitudinally tracking the same neonatal endothelial cells (ECs) over days to weeks, we found that capillary plexus expansion is driven by network-wide vessel regression and transient angiogenesis. A fixed number of neonatal ECs rearrange their positions to evenly distribute throughout the developing plexus and become positionally stable in adulthood. Upon injury, while neonatal ECs are predisposed to die, adult ECs survive through a plasmalemmal self-repair response. Furthermore, adult neighboring ECs reactivate migration to assist vessel repair. Lastly, neonatal vessel regression and adult vascular maintenance are orchestrated by temporally restricted VEGFR2 dependent signaling. Our work sheds light on fundamental cellular mechanisms that underlie both vascular maturation and adult homeostasis in vivo.

developmental biology↗

bioRxiv2022

Characterization of protein isoform diversity in human umbilical vein endothelial cells (HUVECs) via long-read proteogenomics

Endothelial cells (ECs) comprise the lumenal lining of all blood vessels and are critical for the functioning of the cardiovascular system. Their phenotypes can be modulated by protein isoforms. To characterize the isoform landscape within ECs, we applied a long read proteogenomics approach to analyze human umbilical vein endothelial cells (HUVECs). Transcripts delineated from PacBio sequencing serve as the basis for a sample-specific protein database used for downstream MS analysis to infer protein isoform expression. We detected 53,836 transcript isoforms from 10,426 genes, with 22,195 of those transcripts being novel. Furthermore, the predominant isoform in HUVECs does not correspond with the accepted "reference isoform" 25% of the time, with vascular pathway-related genes among this group. We found 2,597 protein isoforms supported through unique peptides, with an additional 2,280 isoforms nominated upon incorporation of long-read transcript evidence. We characterized a novel alternative acceptor for endothelial-related gene CDH5, suggesting potential changes in its associated signaling pathways. Finally, we identified novel protein isoforms arising from a diversity of splicing mechanisms supported by uniquely mapped novel peptides. Our results represent a high resolution atlas of known and novel isoforms of potential relevance to endothelial phenotypes and function. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/490813v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@86ca88org.highwire.dtl.DTLVardef@15ab038org.highwire.dtl.DTLVardef@e7553borg.highwire.dtl.DTLVardef@18cbae8_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2022

In vitro engineering of the lung alveolus

Therapeutic lung regeneration is predicated upon successful reconstitution of lung alveoli, the functional units of gas exchange. Here, we identify requisite multimodal cues that are critical to reconstructing the alveolar epithelium and alveoli in lung scaffolds. Alveolar reconstruction in vitro is divided into several distinct phases. In the first phase, endothelial cells coordinate with fibroblasts and select exogenous factors to promote alveolar scaffold population with surfactant-secreting alveolar epithelial type 2 cells (AEC2s). After formation of organized epithelial alveolar-like structures, subsequent withdrawal of Wnt and FGF agonism synergizes with tidal-level mechanical strain to induce differentiation of AEC2s to squamous type 1 AECs (AEC1s) in cultured alveoli, in situ. These results outline a rational strategy to engineer an alveolus of AEC2s and AEC1s contained within epithelial-mesenchymal-endothelial units, and reveal the critical interplay amongst biochemical, cellular, and mechanical niche cues within the reconstituting alveolus.

bioengineering↗

bioRxiv2022

Connexin 43-mediated Neurovascular Interactions Regulate Neurogenesis in the Adult Brain Subventricular Zone

The subventricular zone (SVZ) is the largest neural stem cell (NSC) niche in the adult brain; herein, the blood-brain barrier is leaky, allowing direct interactions between NSCs and endothelial cells (ECs). Mechanisms by which direct NSC-EC interactions in the SVZ control NSC behavior are unclear. We found Cx43 to be highly expressed by both NSCs and ECs in the SVZ, and its deletion in either cell type leads to increased NSC proliferation and neuroblast generation, suggesting that Cx43-mediated NSC-EC interactions maintain NSC quiescence. This is further supported by in vitro studies showing co-culture with ECs decreases NSC proliferation and increases their expression of genes associated with quiescence in a Cx43-dependent manner. Cx43 mediates these effects in a channel-independent manner involving its cytoplasmic tail and ERK activation. Such insights further advance our understanding of NSC regulation in vivo and may inform NSC maintenance ex vivo for stem cell therapies for neurodegenerative disorders.

neuroscience↗

bioRxiv2022

Transformation of alignment files improves performance of variant callers for long-read RNA sequencing data

Long-read RNA sequencing (lrRNA-seq) produces detailed information about full-length transcripts, including novel and sample-specific isoforms. Furthermore, there is opportunity to call variants directly from lrRNA-seq data. However, most state-of-the-art variant callers have been developed for genomic DNA. Here, there are two objectives: first, we perform a mini-benchmark on GATK, DeepVariant, Clair3, and NanoCaller primarily on PacBio Iso-Seq, data, but also on Nanopore and Illumina RNA-seq data; second, we propose a pipeline to process spliced-alignment files, making them suitable for variant calling with DNA-based callers. With such manipulations, high calling performance can be achieved using DeepVariant on Iso-seq data.

bioinformatics↗

bioRxiv2021

Distinct Hypoxia-induced Translational Profiles of Embryonic and Adult-derived Macrophages

Tissue homeostasis and repair are orchestrated by resident and newly recruited macrophages that alter their gene expression program in response to changes in tissue microenvironment. Embryonic macrophages, such as fetal liver derived macrophages (FLDM) seed the organs, including heart and lung during embryonic development and persist throughout the adult lifetime, while bone marrow-derived macrophages (BMDM) are recruited following an acute perturbation. Transcriptome analyses of FLDM and BMDM identified differences between them at the level of RNA expression, which correlates imperfectly with protein levels. Post-transcriptional regulation by microRNAs (miRNAs) and RNA-binding proteins determines mRNA stability and translation rate and may override transcriptional cues in response to environmental changes, such as hypoxia. To identify distinct features of FLDM and BMDM response to hypoxia at the level of translation, we employed translating ribosome affinity purification (TRAP) to isolate polysomal RNA. RNA-seq profiling of translated RNA identified distinct hypoxia-induced translational signature of BMDM (Ly6e, vimentin and glycolysis-associated enzymes Pgk1, Tpi1, Aldoa, Ldha) and FLDM (chemokines Ccl7 and Ccl2). By translational profiling of BMDM and FLDM with deletion of the RNA-binding protein HuR, we identified transcripts that were dependent on HuR. These findings highlight the importance of HuR and identify its distinct targets for post-transcriptional regulation of gene expression in embryonic vs. adult-derived macrophages.

cell biology↗

bioRxiv2021

The Cell Surface Receptors Ror1/2 Control Cardiac Myofibroblast Differentiation

BackgroundA hallmark of heart failure is cardiac fibrosis, which results from the injury-induced differentiation response of resident fibroblasts to myofibroblasts that deposit extracellular matrix. During myofibroblast differentiation, fibroblasts progress through polarization stages of early pro-inflammation, intermediate proliferation, and late maturation, but the regulators of this progression are poorly understood. Planar cell polarity receptors, receptor tyrosine kinase like orphan receptor 1 and 2 (Ror1/2), can function to promote cell differentiation and transformation. In this study, we investigated the role of the Ror1/2 in a model of heart failure with emphasis on myofibroblast differentiation. Methods and ResultsThe role of Ror1/2 during cardiac myofibroblast differentiation was studied in cell culture models of primary murine cardiac fibroblast activation and in knockout mouse models that underwent transverse aortic constriction (TAC) surgery to induce cardiac injury by pressure overload. Expression of Ror1 and Ror2 were robustly and exclusively induced in fibroblasts in hearts after TAC surgery, and both were rapidly upregulated after early activation of primary murine cardiac fibroblasts in culture. Cultured fibroblasts isolated from Ror1/2-KO mice displayed a pro-inflammatory phenotype indicative of impaired myofibroblast differentiation. Although the combined ablation of Ror1/2 in mice did not result in a detectable baseline phenotype, TAC surgery led to the death of all mice by day 6 that was associated with myocardial hyper-inflammation and vascular leakage. ConclusionsTogether, these results show that Ror1/2 are essential for the progression of myofibroblast differentiation and for the adaptive remodeling of the heart in response to pressure overload.

cell biology↗

bioRxiv2020

Endothelial Cell Cycle State Determines Propensity for Arterial-Venous Fate

Formation and maturation of a functional blood vascular system is required for the development and maintenance of all tissues in the body. During the process of blood vessel development, primordial endothelial cells are formed and become specified toward arterial or venous fates to generate a circulatory network that provides nutrients and oxygen to, and removes metabolic waste from, all tissues1-3. Specification of arterial and venous endothelial cells occurs in conjunction with suppression of endothelial cell cycle progression4,5, and endothelial cell hyperproliferation is associated with potentially lethal arterial-venous malformations6. However, the mechanistic role that cell cycle state plays in arterial-venous specification is unknown. Herein, studying retinal vascular development in Fucci2aR reporter mice7, we found that venous and arterial endothelial cells are in distinct cell cycle states during development and in adulthood. That is, venous endothelial cells reside in early G1 state, while arterial endothelial cells reside in late G1 state. Endothelial cells in early vs. late G1 exhibited significant differences in gene expression and activity, especially among BMP/TGF-{beta} signaling components. The early G1 state was found to be essential for BMP4-induced venous specification, whereas late G1 state is essential for TGF-{beta}1-induced arterial specification. In a mouse model of endothelial cell hyperproliferation and disrupted vascular remodeling, pharmacological inhibition of endothelial cell cycle rescues the arterial-venous specification defects. Collectively, our results show that endothelial cell cycle control plays a key role in arterial-venous network formation, and distinct cell cycle states provide distinct windows of opportunity for the molecular induction of arterial vs. venous specification.

developmental biology↗