Probes for INS

Hyaluronan (HA) plays an important role in the development and maintenance of tissues, and its degradation is implicated in many pathologic conditions. We recently reported that HA-binding protein involved in HA depolymerization (HYBID/KIAA1199; encoded by CEMIP) is a key molecule in HA depolymerization, but its developmental and pathologic functions remain elusive. We generated Hybid-deficient mice using the Cre/locus of crossover in P1 (loxP) system and analyzed their phenotypes. Hybid-deficient mice were viable and fertile, but their adult long bones were shorter than those of wild-type animals. Hybid-deficient mice showed lengthening of hypertrophic zone in the growth plate until 4 weeks after birth. There were fewer capillaries and osteoclasts at the chondroosseous junction in the Hybid-deficient mice compared with the wild-type mice. In situ hybridization demonstrated that Hybid was expressed by hypertrophic chondrocytes at the chondroosseous junction. Cultured primary chondrocytes expressed higher levels of Hybid than did osteoblasts or osteoclasts, and the Hybid expression in the chondrocytes was up-regulated after maturation to hypertrophic chondrocytes. High-molecular-weight HA was accumulated in the lengthened hypertrophic zone in Hybid-deficient mice. In addition, high-molecular-weight HA significantly reduced cell growth and tube formation in vascular endothelial growth factor-stimulated or -nonstimulated endothelial cells. HA metabolism by HYBID is involved in endochondral ossification during postnatal development by modulation of angiogenesis and osteoclast recruitment at the chondroosseous junction.

Deficiency of vascular endothelial growth factor A (VEGF) has been associated with severe craniofacial anomalies in both humans and mice. Cranial neural crest cell (NCC)-derived VEGF regulates proliferation, vascularization and ossification of cartilage and membranous bone. However, the function of VEGF derived from specific subpopulations of NCCs in controlling unique aspects of craniofacial morphogenesis is not clear. In this study a conditional knockdown strategy was used to genetically delete Vegfa expression in Osterix (Osx) and collagen II (Col2)-expressing NCC descendants. No major defects in calvaria and mandibular morphogenesis were observed upon knockdown of VEGF in the Col2+ cell population. In contrast, loss of VEGF in Osx+ osteoblast progenitor cells led to reduced ossification of calvarial and mandibular bones without affecting the formation of cartilage templates in newborn mice. The early stages of ossification in the developing jaw revealed decreased initial mineralization levels and a reduced thickness of the collagen I (Col1)-positive bone template upon loss of VEGF in Osx+ precursors. Increased numbers of proliferating cells were detected within the jaw mesenchyme of mutant embryos. Explant culture assays revealed that mandibular osteogenesis occurred independently of paracrine VEGF action and vascular development. Reduced VEGF expression in mandibles coincided with increased phospho-Smad1/5 (P-Smad1/5) levels and bone morphogenetic protein 2 (Bmp2) expression in the jaw mesenchyme. We conclude that VEGF derived from Osx+ osteoblast progenitor cells is required for optimal ossification of developing mandibular bones and modulates mechanisms controlling BMP-dependent specification and expansion of the jaw mesenchyme.

The International Endocervical Adenocarcinoma Criteria and Classification was developed to separate endocervical adenocarcinomas (ECAs) into 2 main categories on the basis of morphology such as human papilloma virus-associated (HPVA) and non-human papilloma virus-associated adenocarcinomas. We aimed to improve the diagnostic accuracy of International Endocervical Adenocarcinoma Criteria and Classification by performing a comprehensive immunohistochemical evaluation and constructing objective immunohistochemical-based algorithms for the classification of these tumors. Tissue microarrays were constructed from 297 of 409 cases used to develop the original classification. Immunostains included p16, p53, estrogen receptor (ER), progesterone receptor, androgen receptor, Vimentin, CK7, CK20, HER2, HIK1083, MUC6, CA-IX, SATB2, HNF-1beta, napsin A, PAX8, CDX2, GATA3, p63, p40, and TTF-1. High-risk human papilloma virus (HR-HPV) was detected by in situ hybridization (ISH) using probes against E6 and E7 mRNA expressed in 18 different virus types. Vimentin, ER, and progesterone receptor were expressed in a significant minority of ECAs, mostly HPVAs, limiting their use in differential diagnosis of endometrioid carcinoma when unaccompanied by HPV-ISH or p16. HR-HPV ISH had superior sensitivity, specificity, and negative and positive predictive values compared with p16, as published previously. HNF-1beta did not have the anticipated discriminatory power for clear cell carcinoma, nor did MUC6 or CA-IX for gastric-type carcinoma. HNF-1beta and napsin A were variably expressed in clear cell carcinoma, with HNF-1beta demonstrating less specificity, as it was ubiquitously expressed in gastric-type carcinoma and in the majority of HPV-associated mucinous (predominantly intestinal-type and invasive ECA resembling stratified mucin-producing intraepithelial lesion [iSMILE]) and usual-type carcinomas. HIK1083 was expressed in nearly half of gastric-type carcinomas, but not in the vast majority of other subtypes. GATA3 was positive in 10% of usual-type adenocarcinomas and in single examples of other subtypes. Rare gastric-type and HPVA mucinous carcinomas displayed HER2 overexpression. Androgen receptor was positive in 6% of usual-type adenocarcinomas. Aberrant p53 expression was found in only 3.6% of usual-type HPVA carcinomas, but it was more prevalent in mucinous (intestinal type and iSMILE) HPVAs and non-human papilloma virus-associates (particularly in gastric-type carcinoma, >50% of cases). The following diagnostic classification algorithms were developed with the above data. Carcinomas without overt cytoplasmic mucin (endometrioid, usual-type endocervical, clear cell, and mesonephric carcinomas) can be subclassified using HR-HPV ISH, ER, and GATA3, whereas carcinomas with easily appreciated cytoplasmic mucin (endometrioid carcinoma with mucinous features, HPVA mucinous, and gastric-type carcinomas) can be subclassified with HR-HPV ISH and ER.

Gastric cancer is the fifth most common cancer and third leading cause of cancer-related death worldwide. Several studies on angiogenic blocking agents in gastric cancer revealing promising results by the use of monoclonal antibodies against VEGFA or its receptor VEGFR2 or against VEGFA activating pathway. The validation of biomarkers useful to better organize the clinical trials involving anti-angiogenic therapies is crucial. Molecular markers such as RNA are increasingly used for cancer diagnosis, prognosis, and therapy guidance as in the case of the targeted therapies concerning the inhibition of angiogenesis. The aim of this study is to set the conditions for evaluating the expression of VEGFA and VEGFR2 in gastric cancer specimens and in healthy gastric mucosa by the use of RNAscope, a novel RNA in situ hybridization (ISH) method that allows the visualization of a specific gene expression in individual cells. We found the increased expression of VEGFA in the tubular glands and VEGFR2 in the endothelium of gastric cancer samples mainly in the T2, T3 and T4 stages of tumor progression as compared to the healthy controls. These results obtained by the application of this highly sensitive method for oligonucleotide detection the role of angiogenesis in gastric cancer progression already highlighted by conventional immunohistochemical methods, and offer significant promise as a new platform for developing and implementing RNA-based molecular diagnostics also in the conditions in which immunohistochemistry is not applicable.

Amplification of vascular endothelial growth factor A (VEGFA) has been recently reported in TFEB-amplified renal cell carcinomas regardless the level of TFEB amplification. We sought to determine VEGFA amplification by fluorescent in situ hybridization (FISH) and VEGFA mRNA expression by in situ hybridization (RNAscope 2.5) in a series of 10 renal cell carcinomas with TFEB gene alterations, either amplification and/or rearrangement (t(6;11) renal cell carcinoma). TFEB gene rearrangement was demonstrated in eight cases, whereas the remaining two cases showed a high level of TFEB (> 10 copies of fluorescent signals) gene amplification without evidence of rearrangement. Among the eight t(6;11) renal cell carcinomas (TFEB-rearranged cases), one case displayed a high level of TFEB gene amplification and two showed increased TFEB gene copy number (3-4 copies of fluorescent signals). Those three cases behaved aggressively. By FISH, VEGFA was amplified in all three cases with TFEB amplification and increased VEGFA gene copy number was observed in the two aggressive cases t(6;11) renal cell carcinomas with an overlapping increased number of TFEB fluorescent signals. Overall, VEGFA mRNA expression was observed in 8 of 10 cases (80%); of these 8 cases, 3 cases showed high-level TFEB amplification, one case showed TFEB rearrangement with increased TFEB gene copy number, whereas four showed TFEB gene rearrangement without increased copy number. In summary, VEGFA amplification/increased gene copy number and VEGFA mRNA expression occur in TFEB-amplified renal cell carcinoma, but also in a subset of t(6;11) renal cell carcinoma demonstrating aggressive behavior, and in unamplified conventional t(6;11) renal cell carcinoma suggesting VEGFA as potential therapeutic target in these neoplasms even in the absence of TFEB amplification. We finally propose that all the renal tumors showing morphological characteristics suggesting t(6;11) renal cell carcinoma and all unclassified renal cell carcinomas, either high grade or low grade, should immunohistochemically be evaluated for cathepsin K and/or Melan-A and if one of them is positive, tested for TFEB gene alteration and VEGFA gene amplification.

Angiostasis mediated by IFNγ is a key mechanism of anti-tumor immunity; however, the effect of IFNγ on host VEGFA-expressing cells during tumor progression is still elusive. Here, we developed transgenic mice with IFNγ receptor (IFNγR) expression under control of the Vegfa promoter (V-γR). In these mice, the IFNγ responsiveness of VEGFA -expressing cells led to a dramatic growth suppression of transplanted lung carcinoma cells. Surprisingly, increased mortality and tumor metastasis were observed in the tumor-bearing V-γR mice, in comparison to the control wild type and IFNγR-deficient mice. Further study showed that perivascular cells were VEGFA-expressing cells and potential IFNγ targets. In vivo, tumor vascular perfusion and pericyte association with blood vessels were massively disrupted in V-γR mice. In vitro, IFNγ inhibited TGF-β-signaling through upregulating SMAD7 and therefore, down-regulated N-cadherin expression in pericytes. Importantly, IFNγ neutralization in vivo using a monoclonal antibody reduced tumor metastasis. Together, the results suggest that IFNγR-mediated dissociation of perivascular cells from blood vessels contributes to the acceleration of tumor metastasis. Thus the inhibition of tumor growth via IFNγ-induced angiostasis might also accelerate tumor metastasis.

Capable of mediating efficient transfection and protein production without eliciting innate immune responses, chemically modified mRNA holds great potential to produce paracrine factors at a physiologically beneficial level, in a spatiotemporally controlled manner, and with low toxicity. Although highly promising in cardiovascular medicine and wound healing, effects of this emerging therapeutic on the microvasculature and its bioactivity in disease settings remain poorly understood. Here, we longitudinally and comprehensively characterize microvascular responses to AZD8601, a modified mRNA encoding vascular endothelial growth factor A (VEGF-A), in vivo. Using multi-parametric photoacoustic microscopy, we show that intradermal injection of AZD8601 formulated in a biocompatible vehicle results in pronounced, sustained and dose-dependent vasodilation, blood flow upregulation, and neovessel formation, in striking contrast to those induced by recombinant human VEGF-A protein, a non-translatable variant of AZD8601, and citrate/saline vehicle. Moreover, we evaluate the bioactivity of AZD8601 in a mouse model of diabetic wound healing in vivo. Using a boron nanoparticle-based tissue oxygen sensor, we show that sequential dosing of AZD8601 improves vascularization and tissue oxygenation of the wound bed, leading to accelerated re-epithelialization during the early phase of diabetic wound healing.