Probes for INS

Heart failure (HF) is a debilitating disease affecting >64 million people worldwide. In addition to impaired cardiovascular performance and associated systemic complications, most patients with HF suffer from depression and substantial cognitive decline. Although neuroinflammation and brain hypoperfusion occur in humans and rodents with HF, the underlying neuronal substrates, mechanisms, and their relative contribution to cognitive deficits in HF remains unknown.To address this critical gap in our knowledge, we used a well-established HF rat model that mimics clinical outcomes observed in the human population, along with a multidisciplinary approach combining behavioral, electrophysiological, neuroanatomical, molecular and systemic physiological approaches.Our studies support neuroinflammation, hypoperfusion/hypoxia, and neuronal deficits in the hippocampus of HF rats, which correlated with the progression and severity of the disease. An increased expression of AT1aRs (Ang [angiotensin] II receptor type 1a) in hippocampal microglia preceded the onset of neuroinflammation. Importantly, blockade of AT1Rs with a clinically used therapeutic drug (Losartan), and delivered in a clinically relevant manner, efficiently reversed neuroinflammatory end points (but not hypoxia ones), resulting in turn in improved cognitive performance in HF rats. Finally, we show than circulating Ang II can leak and access the hippocampal parenchyma in HF rats, constituting a possible source of Ang II initiating the neuroinflammatory signaling cascade in HF.In this study, we identified a neuronal substrate (hippocampus), a mechanism (Ang II-driven neuroinflammation) and a potential neuroprotective therapeutic target (AT1aRs) for the treatment of cognitive deficits in HF.

Although lymph node (LN) metastasis is an important prognostic parameter in cervical cancer, the tissue remodeling at a pre-metastatic state is poorly documented in LNs. We here identified periostin (POSTN) as a component of non-metastatic LNs by applying proteomic analyses and computerized image quantifications on LNs of patients with cervical cancer. We provide evidence for remarkable modifications of POSTN and lymphatic vessel distributions and densities in non-metastatic sentinel and metastatic human LNs, when compared to distant non-metastatic LNs. POSTN deposition at a pre-metastatic stage was demonstrated in a pre-clinical murine model (the ear sponge assay). Its expression by fibroblastic LN cells was assessed by in situ hybridization and in vitro cultures. In vitro, POSTN promoted lymphatic endothelial cell functions and tumor cell proliferation. Accordingly, the in vivo injection of recombinant POSTN together with VEGF-C boosted the lymphangiogenic response, while the metastatic potential of tumor cells was drastically reduced using a POSTN blocking antibody. This translational study also supports the existence of an unprecedented dialog "in cascade", between the primary tumor and the first pelvic nodal relay in early cervical cancer, and subsequently from pelvic LN to para-aortic LNs in locally advanced cervical cancers. Collectively, this work highlights the association of POSTN deposition with lymphangiogenesis in LNs, and provides evidence for a key contribution of POSTN in promoting VEGF-C driven lymphangiogenesis and the seeding of metastatic cells.

Plasma IL-6 is elevated after myocardial infarction (MI) and is associated with increased morbidity and mortality. Which cardiac cell type preferentially contributes to IL-6 and how its production is regulated is largely unknown. Here, we studied the cellular source and purinergic regulation of IL-6 formation in a murine MI model. IL-6, measured in various cell types in post MI hearts by qPCR, RNAscope and at protein level, was preferentially formed by fibroblasts (CFs). scRNAseq in infarcted mouse and human hearts confirmed this finding. Adenosine stimulated fibroblast IL-6 formation via A2bR in a Gq-dependent manner. CFs highly expressed Adora2b, rapidly degraded extracellular ATP to AMP but lacked CD73. In mice and humans Adora2B was also mainly expressed by fibroblasts (scRNAseq). Global IL-6 formation was assessed in isolated hearts in mice lacking CD73 on T-cells (CD4CD73-/-) a condition known to be associated with adverse cardiac remodeling. The ischemia-induced release of IL-6 was strongly attenuated in CD4CD73-/- mice, suggesting adenosine-mediated modulation. Together this demonstrates that post-MI IL-6 is mainly derived from activated CFs and is controlled by T-cell derived adenosine. Purinergic metabolic cooperation between CFs and T-cells is a novel mechanism with therapeutic potential which modulates IL6 formation by the heart.

The arcuate nucleus (ArcN) is an integrative hub for the regulation of energy balance, reproduction, and arterial pressure (AP), all of which are influenced by Angiotensin II (AngII); however, the cellular mechanisms and downstream neurocircuitry are unclear. Here we show that ArcN AngII increases AP in female rats via two phases, both of which are mediated via activation of AngII type 1 receptors (AT1aR): initial vasopressin-induced vasoconstriction, followed by slowly developing increases in sympathetic nerve activity (SNA) and heart rate (HR). In male rats, ArcN AngII evoked a similarly slow increase in SNA, but the initial pressor response was variable. In females, the effects of ArcN AngII varied during the estrus cycle, with significant increases in SNA, HR, and AP occurring during diestrus and estrus, but only increased AP during proestrus. Pregnancy markedly increased the expression of AT1aR in the ArcN with parallel substantial AngII-induced increases in SNA and MAP. In both sexes, the sympathoexcitation relied on suppression of tonic ArcN sympathoinhibitory Neuropeptide Y inputs, and activation of pro-opiomelanocortin (POMC) projections, to the paraventricular nucleus (PVN). Few or no NPY or POMC neurons expressed the AT1aR, suggesting that AngII increases AP and SNA at least in part indirectly via local interneurons, which express tyrosine hydroxylase (TH) and VGat (i.e. GABAergic). ArcN TH neurons release GABA locally, and central AT1aR and TH neurons mediate stress responses; therefore, we propose that TH AT1aR neurons are well situated to locally coordinate the regulation of multiple modalities within the ArcN in response to stress.SIGNIFICANCEThe arcuate nucleus (ArcN) is an integrative hub for the regulation of energy balance, reproduction, and arterial pressure (AP), all of which are influenced by Angiotensin II (AngII). Here we show that ArcN AngII activates AT1aR to increase AP in male and female rats by slowly increasing sympathetic nerve activity. In females, ArcN AngII also evoked an initial pressor response mediated by vasopressin-induced vasoconstriction. Pregnant and estrus females responded more than males, in association with higher ArcN AT1aR expression. AT1aR were identified in ArcN interneurons that express tyrosine hydroxylase (TH) and GABA. Since brain AT1aR and TH mediate stress responses, ArcN AT1aR TH neurons are well situated to locally coordinate autonomic, hormonal, and behavioral responses to stress.

The cardiac vascular and perivascular niche are of major importance in homeostasis and during disease, but we lack a complete understanding of its cellular heterogeneity and alteration in response to injury as a major driver of heart failure. Using combined genetic fate tracing with confocal imaging and single-cell RNA sequencing of this niche in homeostasis and during heart failure, we unravel cell type specific transcriptomic changes in fibroblast, endothelial, pericyte and vascular smooth muscle cell subtypes. We characterize a specific fibroblast subpopulation that exists during homeostasis, acquires Thbs4 expression and expands after injury driving cardiac fibrosis, and identify the transcription factor TEAD1 as a regulator of fibroblast activation. Endothelial cells display a proliferative response after injury, which is not sustained in later remodeling, together with transcriptional changes related to hypoxia, angiogenesis, and migration. Collectively, our data provides an extensive resource of transcriptomic changes in the vascular niche in hypertrophic cardiac remodeling.

The renin-angiotensin system (RAS) is an essential hormonal system involved in water and sodium reabsorption, renal blood flow regulation, and arterial constriction. Systemic stimulation of the RAS with infusion of the main peptide angiotensin II (Ang II) in animals as well as pathological elevation of renin (i.e renovascular hypertension) to increase circulatory Ang II in humans ultimately lead to hypertension and end-organ damage. In addition to hypertension, accumulating evidence support that the Ang II type 1 receptor exerts a critical role in cardiovascular and kidney diseases independent of blood pressure elevation. In the last two decades, the identification of an increased number of peptides and receptors has facilitated the concept that the RAS has both detrimental and beneficial effects on the cardiovascular system depending on which RAS components are activated. For example, angiotensin 1-7 and Ang II type 2 receptors act as a counter-regulatory system against the classical RAS by mediating vasodilation. While the RAS as an endocrine system for regulation of blood pressure is well established, there remain many unanswered questions and controversial findings regarding blood pressure regulation and pathophysiological regulation of cardiovascular diseases at the tissue level. This review article will include the latest knowledge gleaned from cell type-selective gene deleted mice regarding cell type-specific roles of AngII receptors and discuss their significance in health and diseases. In particular, we focus on the roles of these receptors expressed in vascular, cardiac, and kidney epithelial cells.

Large joints are composed of two closely linked cartilages: articular cartilage (AC; rich in type II collagen, a well-studied tissue) and fibrocartilaginous enthesis (FE; rich in type I collagen, common disorder sites of enthesopathy and sporting injuries, although receiving little attention). For many years, both cartilages were thought to be formed by chondrocytes, whereas tendon, which attaches to the humeral bone head, is primarily considered as a completely different connective tissue. In this study, we raised an unconventional hypothesis: tendon cells directly form FE via cell transdifferentiation. To test this hypothesis, we first qualitatively and quantitatively demonstrated distinct differences between AC and FE in cell morphology and cell distribution, mineralization status, extracellular matrix (ECM) contents, and critical ECM protein expression profiles using comprehensive approaches. Next, we traced the cell fate of tendon cells using ScxLin (a tendon specific Cre ScxCreERT2; R26R-tdTomato line) with one-time tamoxifen induction at early (P3) or young adult (P28) stages and harvested mice at different development ages, respectively. Our early tracing data revealed different growth events in tendon and FE: an initial increase but gradual decrease in the ScxLin tendon cells and a continuous expansion in the ScxLin FE cells. The young adult tracing data demonstrated continuous recruitment of ScxLin cells into FE expansion during P28 and P56. A separate tracing line, 3.2 Col 1Lin (a so-called "bone-specific" line), further confirmed the direct contribution of tendon cells for FE cell formation, which occurred in days but FE ECM maturation (including high levels of SOST, a potent Wnt signaling inhibitor) took weeks. Finally, loss of function data using diphtheria toxin fragment A (DTA) in ScxLin cells demonstrated a significant reduction of ScxLin cells in both tendons and FE cells, whereas the gain of function study (by stabilizing β-catenin in ScxLin tendon cells via one-time injection of tamoxifen at P3 and harvesting at P60) displayed great expansion of both ScxLin tendon and FE mass. Together, our studies demonstrated that fibrocartilage is an invaded enthesis likely originating from the tendon via a quick cell transdifferentiation mechanism with a lengthy ECM maturation process. The postnatally formed fibrocartilage roots into existing cartilage and firmly connects tendon and bone instead of acting as a simple attachment site as widely believed. We believe that this study will stimulate more intense exploring in this understudied area, especially for patients with enthesopathy and sporting injuries.

PURPOSE: Up to one third of ovarian cancer patients are intrinsically resistant to platinum-based treatment. However, predictive and therapeutic strategies are lacking due to a poor understanding of the underlying molecular mechanisms. This study aimed to identify key molecular characteristics that are associated with primary chemoresistance in epithelial ovarian cancers. EXPERIMENTAL DESIGN: Gene expression profiling was performed on a discovery set of 85 ovarian tumors with clinically well-defined response to chemotherapies as well as on an independent validation data set containing 138 ovarian patients from the chemo-treatment arm of ICON7 trial. RESULTS: We identified a distinct "reactive stroma" gene signature that is specifically associated with primary chemoresistant tumors and was further upregulated in post-treatment recurrent tumors. IHC and RNA ISH analyses on three of the highest-ranked signature genes (POSTN, LOX and FAP) confirmed that modulation of the reactive stroma signature genes within the peritumoral stromal compartments was specifically associated with the clinical chemoresistance. Consistent with these findings, chemosensitive ovarian cells grown in the presence of recombinant POSTN promoted resistance to carboplatin and paclitaxel treatment in vitro. Finally, we validated the reactive stroma signature in an independent dataset and demonstrated that high POSTN expression level predicts shorter progression free survival following front line chemotherapy. CONCLUSIONS: Our findings highlighted the important interplay between cancer and the tumor microenvironment in ovarian cancer biology and treatment. The identified reactive stromal components in this study provided a molecular basis to the further development of novel diagnostic and therapeutic strategies for overcoming chemoresistance in ovarian cancer.

The action of leptin in brain to increase sympathetic nerve activity (SNA) and blood pressure depends upon functional Angiotensin II (AngII) type 1a receptors (AT1aR); however, the sites and mechanism of interaction are unknown. Here we identify one site, the hypothalamic arcuate nucleus (ArcN), since prior local blockade of AT1aR in the ArcN with losartan or candesartan in anesthetized male rats essentially eliminated the sympathoexcitatory and pressor responses to ArcN leptin nanoinjections. Unlike mice, in male and female rats, AT1aR and LepR rarely co-localized, suggesting that this interdependence occurs indirectly, via a local interneuron or network of neurons. ArcN leptin increases SNA by activating pro-opiomelanocortin (POMC) inputs to the PVN, but this activation requires simultaneous suppression of tonic PVN Neuropeptide Y (NPY) sympathoinhibition. Because AngII-AT1aR inhibits ArcN NPY neurons, we propose that loss of AT1aR suppression of NPY blocks leptin-induced increases in SNA; in other words, ArcN-AngII-AT1aR is a gatekeeper for leptin-induced sympathoexcitation. With obesity, both leptin and AngII increase; therefore, the increased AT1aR activation could open the gate, allowing leptin (and insulin) to drive sympathoexcitation unabated, leading to hypertension.

Perturbations in the transcriptional programs specifying epidermal differentiation cause diverse skin pathologies ranging from impaired barrier function to inflammatory skin disease. However, the global scope and organization of this complex cellular program remain undefined. Here we report single-cell RNA sequencing profiles of 92,889 human epidermal cells from 9 normal and 3 inflamed skin samples. Transcriptomics-derived keratinocyte subpopulations reflect classic epidermal strata but also sharply compartmentalize epithelial functions such as cell-cell communication, inflammation, and WNT pathway modulation. In keratinocytes, ∼12% of assessed transcript expression varies in coordinate patterns, revealing undescribed gene expression programs governing epidermal homeostasis. We also identify molecular fingerprints of inflammatory skin states, including S100 activation in the interfollicular epidermis of normal scalp, enrichment of a CD1C+CD301A+ myeloid dendritic cell population in psoriatic epidermis, and IL1βhiCCL3hiCD14+ monocyte-derived macrophages enriched in foreskin. This compendium of RNA profiles provides a critical step toward elucidating epidermal diseases of development, differentiation, and inflammation.

Pediatric hepatoblastoma is the most common primary liver cancer in infants and children. Studies of hepatoblastoma that focus exclusively on tumor cells demonstrate sparse somatic mutations and a common cell of origin, the hepatoblast, across patients. In contrast to the homogeneity these studies would suggest, hepatoblastoma tumors have a high degree of heterogeneity that can portend poor prognosis. In this study, we use single-cell transcriptomic techniques to analyze resected human pediatric hepatoblastoma specimens, and identify five hepatoblastoma tumor signatures that may account for the tumor heterogeneity observed in this disease. Notably, patient-derived hepatoblastoma spheroid cultures predict differential responses to treatment based on the transcriptomic signature of each tumor, suggesting a path forward for precision oncology for these tumors. In this work, we define hepatoblastoma tumor heterogeneity with single-cell resolution and demonstrate that patient-derived spheroids can be used to evaluate responses to chemotherapy.