Biology subjects

Ramchandran, R.

Publications and source records attributed to Ramchandran, R..

7 recordsLinked to original sources

bioRxiv2025

Decreased endothelial cell retinoic acid signaling accelerates progression of single ventricle pulmonary arteriovenous malformations

BackgroundPulmonary arteriovenous malformations (PAVMs) are vascular complications that universally develop in patients with single ventricle congenital heart disease after Glenn surgery. However, the pathophysiological mechanisms underlying single ventricle PAVMs are poorly understood. To comprehensively evaluate molecular changes post-Glenn, we performed single-cell RNA sequencing (scRNAseq) on rat lung samples after Glenn surgery. MethodsUsing adult Sprague Dawley rats, we performed scRNAseq on unfiltered lung samples 3 weeks after left-sided Glenn or sham surgery. We compared endothelial cell (EC) differentially expressed genes (DEGs) in our model to two mouse models of hereditary hemorrhagic telangiectasia (HHT), a hereditary condition characterized by visceral AVMs. Finally, we modified the vitamin A (Vit A) content of Glenn and sham rat diets and re-assessed PAVM shunting and EC transcriptional differences. ResultsUsing scRNAseq (n=4 Glenn, n=4 sham), we identified 13 transcriptionally distinct lung cell clusters, including 3 EC clusters (general, capillary, lymphatic), with pronounced differences between Glenn and sham in the general EC cluster ([~]17% of genes). Comparison to HHT mouse models confirmed overlap of [~]18% of DEGs, including identification of significantly downregulated genes involved in and regulated by all-trans retinoic acid (ATRA) signaling in all 3 models. Dietary deficiency of Vit A intake, a precursor of ATRA, caused increased PAVM shunting (p<0.01) that was mitigated with excess dietary Vit A intake. Lastly, EC-specific RNAseq identified Vit A diet-induced gene expression differences, including regulation of PI3K signaling. ConclusionsIn this study, we report the novel application of scRNAseq to study mechanisms underlying single ventricle PAVMs in a surgical rat model. We identified multiple dysregulated biological processes in rat lung ECs post-Glenn, including decreased ATRA signaling and conserved gene expression patterns with HHT. Dietary modification of Vit A intake altered post-Glenn shunting and represents a novel potential therapeutic strategy for single ventricle PAVMs and HHT AVMs.

physiology↗

bioRxiv2025

DUSP5 Downregulation in Nucleus Accumbens Core Correlates with Synaptic Plasticity and Cue-Induced Cocaine Reinstatement

The United States is currently facing a drug overdose epidemic, with substance use disorder (SUD) characterized by cyclical phases of drug use, withdrawal, and relapse. The nucleus accumbens core (NAcore), a brain region critical for reward and aversion behaviors, undergoes structural and functional synaptic adaptations in response to chronic drug exposure. These changes, particularly in dopamine D1 receptor-expressing medium spiny neurons (D1-MSNs), are implicated in drug-seeking behaviors and synaptic plasticity. However, the molecular mechanisms underlying these adaptations remain poorly understood. In this study, we investigate the role of dual-specificity phosphatase 5 (DUSP5), an phosphatase known to deactivate extracellular signal-regulated kinase (ERK), in cocaine-induced neuroplasticity. While prior research has linked other DUSP family members to various drugs of abuse, the specific role of DUSP5 in cocaine addiction remains unexplored. We hypothesized that lack of DUSP5 contributes to NAcore synaptic plasticity during cue-induced cocaine reinstatement. To test this, we employed a rat cocaine self-administration model, integrated molecular analyses, and mined publicly available single-cell RNA sequencing data from cocaine-treated NAcore. Our findings aim to elucidate the potential involvement of DUSP5 in cocaine-related synaptic adaptations and behavior, addressing a significant gap in the mechanistic understanding of SUD.

neuroscience↗

bioRxiv2024

Severe traumatic brain injury temporally affects cerebral blood flow, endothelial cell phenotype, and cilia

BackgroundPrevious clinical work suggested that altered cerebral blood flow (CBF) in severe traumatic brain injury (sTBI) correlates with poor executive function and clinical outcome. However, the molecular consequences of altered CBF on endothelial cells (ECs) and their blood flow-sensor organelle called cilia are not known. MethodsWe performed laser speckle contrast imaging, single cell isolation, and single cell RNA sequencing (scRNAseq) after sTBI in a closed skull, linear impact mouse model. Validation of select ciliary target protein changes was performed using flow cytometry. Additionally, in vitro experiments modeled the post-injury hypoxic environment to evaluate the effect on cilia protein ARL13B in human brain microvascular ECs. ResultsWe detected immediate reductions in CBF that were sustained for at least 100 minutes in both impacted and non-impacted sides of the brain. Our scRNAseq data detected heterogeneity in the brain cortex-derived EC cluster and demonstrated that two of five unique EC sub-clusters changed their relative proportions post-sTBI. Consistent with flow changes, we identified multiple genes associated with the fluid shear stress pathway that were significantly differentially expressed in brain ECs post-injury. Also, ECs displayed activation of ischemic pathway as early as day 1 post-injury, and enrichment of hypoxia pathway at day 7 and 28 post- injury. Arl13b ciliary gene expression was lost on day 1 in ECs cluster and remained lost for the entire course of the injury. We validated the loss of cilia protein ARL13B specifically from brain ECs as early as day 1 post-injury and detected the protein in the peripheral blood of the injured mice. We also determined that hypoxia could induce loss of ARL13B protein from cultured ECs. ConclusionsIn severe TBI, blood flow is disrupted in both impacted and non-impacted regions of the brain, creating a hypoxic environment that may influence ciliary gene and protein expression on ECs.

neuroscience↗

bioRxiv2024

SNRK regulates TGFbeta levels in atria to control cardiac fibrosis

Atrial fibrosis is central to the pathology of heart failure (HF) and atrial fibrillation (AF). Identifying precise mechanisms underlying atrial fibrosis will provide effective strategies for clinical intervention. This study investigates a metabolic serine threonine kinase gene, sucrose non-fermenting related kinase (SNRK), that we previously reported to control cardiac metabolism and function. Conditional knockout of Snrk in mouse cardiomyocytes (Snrk cmcKO) leads to atrial fibrosis and subsequently HF. The precise mechanism underlying cardiomyocyte SNRK-driven repression of fibrosis is not known. Here, using mouse, rat, and human tissues, we demonstrate that SNRK expression is high in atria, especially in atrial cardiomyocytes. SNRK expression correlates with lower levels of pro-fibrotic protein transforming growth factor-beta 1 (TGF{beta}1) in the atrial cardiomyocytes. In HL-1 adult immortalized mouse atrial cells, using siRNA approaches, we show that Snrk knockdown cells show more TGF{beta}1 secretion, which was also observed in heart lysates from Snrk cardiac-specific knockout mice in vivo. These effects were exacerbated upon infusion of Angiotensin II. Results from Snrk knockdown cardiomyocytes co-cultured with cardiac fibroblasts suggest that SNRK represses TGF{beta}1 signaling (Smad 2/3) in atrial CMs and prevents paracrine cardiac fibroblast activation (-SMA marker). In conclusion, high SNRK expression in atria regulates cardiac homeostasis, by preventing the release of TGF{beta}1 secretion to block cardiac fibrosis. These studies will assist in developing heart chamber-specific fibrosis therapy for non-ischemic HF and AF.

cell biology↗

bioRxiv2024

Glenn circulation causes early and progressive shunting in a surgical model of pulmonary arteriovenous malformations

BackgroundPulmonary arteriovenous malformations (PAVMs) universally develop in patients with single ventricle congenital heart disease (CHD). Single ventricle PAVMs have been recognized for over 50 years, yet they are poorly understood, and we lack any medical therapies. To improve our understanding of single ventricle PAVM initiation and progression, we developed a surgical rat model of Glenn circulation and characterized PAVM physiology over multiple time points. MethodsUsing adult rats, we performed a left thoracotomy and end-to-end anastomosis of the left superior vena cava to the left pulmonary artery (unilateral Glenn), or sham surgical control. To assess for PAVM physiology in the left lung, we quantified intrapulmonary shunting using two independent methods (bubble echocardiography and fluorescent microsphere injection) at 2 weeks, 2 months, and 6 months. Additionally, we performed arterial blood gas measurements to assess oxygenation and plethysmography to assess ventilation. ResultsWe identified pathologic intrapulmonary shunting by bubble echocardiography as early as 2 weeks post-Glenn surgery, and shunting continued chronically at 2- and 6-months post-Glenn. Shunting also progressed over time, demonstrated by increased shunting of 10{micro}m microspheres at 6 months. Shunting was accompanied by mildly decreased arterial oxygenation, but there were no differences in ventilation as quantified by plethysmography. ConclusionsOur surgical animal model of unilateral Glenn circulation re-creates the clinical condition of single ventricle PAVMs with early and progressive intrapulmonary shunting. This model is poised to characterize single ventricle PAVM pathophysiology and lead to mechanistic and therapeutic discovery. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/588015v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@b1c722org.highwire.dtl.DTLVardef@1889af9org.highwire.dtl.DTLVardef@1767519org.highwire.dtl.DTLVardef@1c230_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2021

Coagulation Factor IIIa (f3a) Knockdown in Zebrafish Leads to Defective Angiogenesis and Mild Bleeding Phenotype

AimTissue factor (TF), an initiator of the extrinsic coagulation pathway, is crucial for embryogenesis, as mice lacking TF are embryonically lethal (E10.5). This lethality may be attributed to defects in vascular development and circulatory failure, suggesting additional roles for TF in embryonic development beyond coagulation. In this study, we characterized the role of one of the TF paralogs (f3a) using a zebrafish model. MethodsTo understand the TF evolution across different species, we performed molecular phylogenetic and sequence homology analysis. The expression of f3a during embryonic developmental stages was determined by RT-PCR. Endothelial-specific transgenic lines of zebrafish (flk1:egfp-NLS/kdrl:mCherry-CAAX) was used to image the vascular development. The role of f3a during embryonic development was investigated by mRNA knockdown using Morpholinos (MO), an antisense-based oligonucleotide strategy. The f3a morphants were examined at 52 hpf for defects in morphological appearance, bleeding, and vascular patterning. ResultsSpatiotemporal expression of f3a by qPCR revealed expression in all developmental stages, suggesting that f3a transcripts are both maternally and zygotically expressed. High expression of f3a from 28 hpf to 36 hpf confirmed the role of in the development of the yolk sac, circulation, and fins. f3a MO-injected embryos showed morphological abnormalities, including shorter body lengths and crooked tails. O-dianisidine staining showed f3a MO-injected embryos exhibited bleeding in the trunk (5.44%) and head (9.52%) regions. Using endothelial-specific transgenic lines of zebrafish (flk1:egfp-NLS/kdrl:mCherry-CAAX), imaging of caudal vein plexus, which forms immediately following the onset of circulation and sprouting, showed a 3-fold decrease in f3a morphants versus controls at 48 hpf, suggesting a potential role for f3a in flow-induced angiogenesis. Conclusionf3a is essential for angiogenesis, in addition to its involvement in hemostasis. Key PointsO_LIf3a knockdown by antisense morpholino oligonucleotide (MO)-approach shows bleeding phenotype. C_LIO_LIf3a is essential for angiogenesis, in addition to its involvement in hemostasis. C_LI

developmental biology↗

bioRxiv2020

RNA-binding protein FXR1 drives cMYC translation by mRNA circularization through eIF4F recruitment in ovarian cancer

BackgroundThe RNA-binding protein FXR1 (fragile X-related protein 1) has been implicated as an important regulator of post-transcriptional changes of mRNAs. However, its role in mRNA circularization and recruitment of eukaryotic translation initiation factors for protein translation remains obscure. Here, we aimed to investigate the molecular mechanisms and potential clinical applications of FXR1 in ovarian cancer growth and progression. MethodsFXR1 copy number variation, mRNA expression, protein levels, and their association with prognosis were determined in clinical datasets. An orthotopic ovarian cancer model and bioluminescence imaging were used for preclinical evaluation of FXR1 in vivo. Reverse phase protein arrays (RPPA) and qPCR arrays were performed to identify FXR1s key targets and downstream effects. SUnSET and polysome profiling were used to determine the translational effects of FXR1. Immunoprecipitation and immunofluorescence were performed to identify the interaction between FXR1 and cMYC mRNA and eIF4F complex. RNA-immunoprecipitation (RIP), RNA electrophoretic mobility shift assays (REMSA), proximity ligation assays (PLA), and biochemical assays were used to identify the specific site on cMYC mRNA to which FXR1 binds to promote mRNA circularization and translation. ResultsWe found that amplification and copy-gain of FXR1 increased the expression of FXR1 mRNA and FXR1 protein in ovarian cancer patients, and these events associated with poor prognosis. We demonstrated that FXR1 binds to AU-rich elements (ARE) within the 3 untranslated region (3UTR) of cMYC. As a consequence, FXR1 binding to cMYC 3UTR leads to the circularization of mRNA and facilitated the recruitment of eukaryotic translation initiation factors (eIFs) to translation start site for improving protein synthesis. ConclusionWe found that FXR1 upregulates a known oncogene, cMYC, by binding to AU-rich elements within the 3UTR, leading to the recruitment of the eIF4F complex for cMYC translation. Our findings uncover a novel mechanism of action of FXR1 in tumorigenesis and provides opportunities to use FXR1 and its downstream effectors as biomarkers or therapeutic targets in ovarian and other cancers.

cancer biology↗