Probes for INS

Fibroblast growth factor (FGF) signaling is essential for many developmental processes and plays a pivotal role in skeletal homeostasis, regeneration and wound healing. FGF signals through one of five tyrosine kinase receptors: Fgfr1a, -1b, -2, -3, -4. To characterize the expression of zebrafish fgfr3 from the larval stage to adulthood, we used RNAscope in situ hybridization on paraffin sections of the zebrafish head. Our study revealed spatial and temporal distribution of fgfr3 transcript in chondrocytes of the head cartilages, osteoblasts involved in bone formation, ventricular zone of the brain, undifferentiated mesenchymal cells of the skin, and lens epithelium of the eye. In general, the expression pattern of zebrafish fgfr3 is similar to the expression observed in higher vertebrates.

Glucagon-like peptide-1 receptor (GLP-1R) agonists, used to treat type 2 diabetes and obesity, reduce rates of myocardial infarction and cardiovascular death. The GLP-1R has been localized to the human sinoatrial node; however, its expression in ventricular tissue remains uncertain. Here we studied GLP-1R expression in the human heart using GLP-1R-directed antisera, quantitative PCR, reverse transcription PCR to detect full length mRNA transcripts, and in situ hybridization. GLP1R mRNA transcripts, encompassing the entire open reading frame, were detected in all four cardiac chambers from 15 hearts at levels approximating those detected in human pancreas. In contrast, cardiac GLP2R expression was relatively lower, whereas cardiac GCGR expression was sporadic and not detected in the left ventricle. GLP1R mRNA transcripts were not detected in RNA from human cardiac fibroblasts, coronary artery endothelial, or vascular smooth muscle cells. Human Brunner's glands and pancreatic islets exhibited GLP-1R-immunopositivity and abundant expression of GLP1R mRNA transcripts by in situ hybridization. GLP1R transcripts were also detected by in situ hybridization in human cardiac sinoatrial node tissue. However definitive cellular localization of GLP1R mRNA transcripts or immunoreactive GLP-1R protein within human cardiomyocytes (CMs) or cardiac blood vessels remained elusive. Moreover, validated GLP-1R antisera lacked sufficient sensitivity to detect expression of the endogenous islet or cardiac GLP-1R by Western blotting. Hence, although human cardiac ventricles express the GLP1R, the identity of one or more ventricular cell type(s) that express a translated GLP1R protein requires further clarification with highly sensitive methods of detection.

The differentiated phenotype of articular chondrocytes of synovial joints needs to be maintained throughout life. Disruption of the articular cartilage, frequently associated with chondrocyte hypertrophy and calcification, is a central feature in osteoarthritis (OA). However, the molecular mechanisms whereby phenotypes of articular chondrocytes are maintained and pathological calcification is inhibited remain poorly understood. Recently, the ecto-enzyme ENPP1, a suppressor of pathological calcification, was reported to be decreased in joint cartilage with OA in both human and mouse, and Enpp1 deficiency causes joint calcification. Here we found that Hedgehog signaling activation contributes to ectopic joint calcification in the Enpp1-/- mice. In the Enpp1-/- joints, Hedgehog signaling was upregulated. Further activation of Hedgehog signaling by removing Patched 1 in the Enpp1-/- mice enhanced ectopic joint calcification, while removing Gli2 partially rescued the ectopic calcification phenotype. Additionally, reduction of Gαs in the Enpp1-/- mice also enhanced joint calcification, suggesting Enpp1 inhibited Hedgehog signaling and chondrocyte hypertrophy by activating Gαs-PKA signaling. Our findings provide new insights in the mechanisms underlying Enpp1 regulation of joint integrity.

Osteoblast-lineage cells in bone human were recently shown to colonize eroded bone surfaces and to closely interact with osteoclasts. They proved identical with reversal cells and are believed to differentiate into bone forming osteoblasts thereby coupling resorption and formation. However, they also exert catabolic activity that contributes to osteoclastic bone resorption, but this has not received much attention. Herein, we used co-cultures of primary human osteoblast-lineage cells and human osteoclasts derived from peripheral blood monocytes to investigate whether a catabolic activity of osteoblast-lineage cells may impact on osteoclastic bone resorption. Through a combination of immunofluorescence, in-situ hybridization, and time-lapse we show that MMP-13 expressing osteoblast-lineage cells are attracted to and closely interact with bone resorbing osteoclasts. This close interaction results in a strong and significant increase in the bone resorptive activity of osteoclasts - especially those making trenches. Importantly, we show that osteoclastic bone resorption becomes sensitive to inhibition of matrix metalloproteinases in the presence, but not in the absence, of osteoblast-lineage cells. We propose that this may be due to the direct action of osteoblast-lineage-derived MMP-13 on bone resorption.

In response to myocardial infarction (MI), cardiac macrophages regulate inflammation and scar formation. We hypothesized that macrophages undergo polarization state changes over the MI time course and assessed macrophage polarization transcriptomic signatures over the first week of MI. C57BL/6 J male mice (3-6 months old) were subjected to permanent coronary artery ligation to induce MI, and macrophages were isolated from the infarct region at days 1, 3, and 7 post-MI. Day 0, no MI resident cardiac macrophages served as the negative MI control. Whole transcriptome analysis was performed using RNA-sequencing on n = 4 pooled sets for each time. Day 1 macrophages displayed a unique pro-inflammatory, extracellular matrix (ECM)-degrading signature. By flow cytometry, day 0 macrophages were largely F4/80highLy6Clow resident macrophages, whereas day 1 macrophages were largely F4/80lowLy6Chigh infiltrating monocytes. Day 3 macrophages exhibited increased proliferation and phagocytosis, and expression of genes related to mitochondrial function and oxidative phosphorylation, indicative of metabolic reprogramming. Day 7 macrophages displayed a pro-reparative signature enriched for genes involved in ECM remodeling and scar formation. By triple in situ hybridization, day 7 infarct macrophages in vivo expressed collagen I and periostin mRNA. Our results indicate macrophages show distinct gene expression profiles over the first week of MI, with metabolic reprogramming important for polarization. In addition to serving as indirect mediators of ECM remodeling, macrophages are a direct source of ECM components. Our study is the first to report the detailed changes in the macrophage transcriptome over the first week of MI.

Oral administration of therapeutic peptides is hindered by poor absorption across the gastrointestinal barrier and extensive degradation by proteolytic enzymes. Here, we investigated the absorption of orally delivered semaglutide, a glucagon-like peptide-1 analog, coformulated with the absorption enhancer sodium N-[8-(2-hydroxybenzoyl) aminocaprylate] (SNAC) in a tablet. In contrast to intestinal absorption usually seen with small molecules, clinical and preclinical dog studies revealed that absorption of semaglutide takes place in the stomach, is confined to an area in close proximity to the tablet surface, and requires coformulation with SNAC. SNAC protects against enzymatic degradation via local buffering actions and only transiently enhances absorption. The mechanism of absorption is shown to be compound specific, transcellular, and without any evidence of effect on tight junctions. These data have implications for understanding how highly efficacious and specific therapeutic peptides could be transformed from injectable to tablet-based oral therapies.

The periodontal complex is essential for tooth attachment and function and includes the mineralized tissues, cementum and alveolar bone, separated by the unmineralized periodontal ligament (PDL). To gain insights into factors regulating cementum-PDL and bone-PDL borders and protecting against ectopic calcification within the PDL, we employed a proteomic approach to analyze PDL tissue from progressive ankylosis knock-out (Ank−/−) mice, featuring reduced PPi, rapid cementogenesis, and excessive acellular cementum. Using this approach, we identified the matrix protein osteopontin (Spp1/OPN) as an elevated factor of interest in Ank−/− mouse molar PDL. We studied the role of OPN in dental and periodontal development and function. During tooth development in wild-type (WT) mice, Spp1 mRNA was transiently expressed by cementoblasts and strongly by alveolar bone osteoblasts. Developmental analysis from 14 to 240 days postnatal (dpn) indicated normal histological structures in Spp1−/− comparable to WT control mice. Microcomputed tomography (micro-CT) analysis at 30 and 90 dpn revealed significantly increased volumes and tissue mineral densities of Spp1−/− mouse dentin and alveolar bone, while pulp and PDL volumes were decreased and tissue densities were increased. However, acellular cementum growth was unaltered in Spp1−/− mice. Quantitative PCR of periodontal-derived mRNA failed to identify potential local compensators influencing cementum in Spp1−/− vs. WT mice at 26 dpn. We genetically deleted Spp1 on the Ank−/− mouse background to determine whether increased Spp1/OPN was regulating periodontal tissues when the PDL space is challenged by hypercementosis in Ank−/− mice. Ank−/−; Spp1−/−double deficient mice did not exhibit greater hypercementosis than that in Ank−/− mice. Based on these data, we conclude that OPN has a non-redundant role regulating formation and mineralization of dentin and bone, influences tissue properties of PDL and pulp, but does not control acellular cementum apposition. These findings may inform therapies targeted at controlling soft tissue calcification.