Quantitative proteomics reveals CLR interactome in primary human cells.

The G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) mediates essential functions in several cell types and is implicated in cardiovascular pathologies, skin diseases, migraine, and cancer. To date, the network of proteins interacting with CLR ("CLR interactome") in primary cells, where this GPCR is expressed at endogenous (physiologically relevant) levels, remains unknown. To address this knowledge gap, we established a novel integrative methodological workflow/approach for conducting a comprehensive/proteome-wide analysis of Homo sapiens CLR interactome. We used primary human dermal lymphatic endothelial cells and combined immunoprecipitation utilizing anti-human CLR antibody with label-free quantitative nano LC-MS/MS and quantitative in situ proximity ligation assay. By using this workflow, we identified 37 proteins interacting with endogenously expressed CLR amongst 4902 detected members of the cellular proteome (by quantitative nano LC-MS/MS) and revealed direct interactions of two kinases and two transporters with this GPCR (by in situ proximity ligation assay). All identified interactors have not been previously reported as members of CLR interactome. Our approach and findings uncover the hitherto unrecognized compositional complexity of the interactome of endogenously expressed CLR and contribute to fundamental understanding of the biology of this GPCR. Collectively, our study provides a first-of-its-kind integrative methodological approach and datasets as valuable resources and robust platform/springboard for advancing the discovery and comprehensive characterization of physiologically relevant CLR interactome at a proteome-wide level in a range of cell types and diseases in future studies.

MEF2 transcription factors are key regulators of sprouting angiogenesis.

Angiogenesis, the fundamental process by which new blood vessels form from existing ones, depends on precise spatial and temporal gene expression within specific compartments of the endothelium. However, the molecular links between proangiogenic signals and downstream gene expression remain unclear. During sprouting angiogenesis, the specification of endothelial cells into the tip cells that lead new blood vessel sprouts is coordinated by vascular endothelial growth factor A (VEGFA) and Delta-like ligand 4 (Dll4)/Notch signaling and requires high levels of Notch ligand DLL4. Here, we identify MEF2 transcription factors as crucial regulators of sprouting angiogenesis directly downstream from VEGFA. Through the characterization of a Dll4 enhancer directing expression to endothelial cells at the angiogenic front, we found that MEF2 factors directly transcriptionally activate the expression of Dll4 and many other key genes up-regulated during sprouting angiogenesis in both physiological and tumor vascularization. Unlike ETS-mediated regulation, MEF2-binding motifs are not ubiquitous to all endothelial gene enhancers and promoters but are instead overrepresented around genes associated with sprouting angiogenesis. MEF2 target gene activation is directly linked to VEGFA-induced release of repressive histone deacetylases and concurrent recruitment of the histone acetyltransferase EP300 to MEF2 target gene regulatory elements, thus establishing MEF2 factors as the transcriptional effectors of VEGFA signaling during angiogenesis.

Calcitonin gene-related peptide and intermedin induce phosphorylation of p44/42 MAPK in primary human lymphatic endothelial cells in vitro.

Calcitonin gene-related peptide (CGRP) and adrenomedullin 2/intermedin (AM2/IMD) play important roles in several pathologies, including cardiovascular disease, migraine and cancer. The efficacy of drugs targeting CGRP signalling axis for the treatment of migraine patients is sometimes offset by side effects (e.g. inflammation and microvascular complications, including aberrant neovascularisation in the skin). Recent studies using animal models implicate CGRP in lymphangiogenesis and lymphatic vessel function. However, whether CGRP or AM2/IMD can act directly on lymphatic endothelial cells is unknown. Here, we found that CGRP and AM2/IMD induced p44/42 MAPK phosphorylation in a time- and dose-dependent manner in primary human dermal lymphatic endothelial cells (HDLEC) in vitro, and thus directly affected these cells. These new findings reveal CGRP and AM2/IMD as novel regulators of LEC biology and warrant further investigation of their roles in the context of pathologies associated with lymphatic function in the skin and other organs, and therapies targeting CGRP signalling axis.

Vascular endothelium in cancer.

The vascular endothelium plays an essential role during organogenesis and in tissue homeostasis. Growing evidence also supports its essential and complex role in tumour biology and cancer progression. In particular, excessive proliferation and transformation or dysfunction of endothelial cells leads to pathological (lymph)angiogenesis or vascular malfunctions, which are hallmarks of neoplastic and malignant disorders. Reciprocal interactions between endothelial cells and the local tumour microenvironment may regulate tumour progression and resistance to anti-cancer therapies in a tumour-type-specific manner.

Kaposi's sarcoma-associated herpesvirus-encoded interleukin-6 and G-protein-coupled receptor regulate angiopoietin-2 expression in lymphatic endothelial cells.

Kaposi's sarcoma (KS) is caused by Kaposi's sarcoma-associated herpesvirus (KSHV) and consists of proliferating spindle cells, which are related to lymphatic endothelial cells (LEC). Angiopoietin-2 (Ang2) is a secreted proangiogenic and lymphangiogenic molecule. Here, we show the expression of Ang2 protein in KS and confirm that KSHV infection up-regulates Ang2 in LEC. We show that a paracrine mechanism contributes to this up-regulation. A lentiviral library of individual KSHV-encoding genes, comprising the majority of known latent genes and a selection of lytic viral genes, was constructed to investigate the underlying mechanism of this up-regulation. Two lytic genes, viral interleukin-6 (vIL6) and viral G-protein-coupled receptor (vGPCR), up-regulated Ang2 expression in LEC. Both vIL6 and vGPCR are expressed in KSHV-infected LEC and caused up-regulation of Ang2 in a paracrine manner. KSHV, vIL6, and vGPCR up-regulated Ang2 through the mitogen-activated protein kinase (MAPK) pathway. Gene expression microarray analysis identified several other angiogenic molecules affected by KSHV, including the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) axis, which is also affected by vIL6 and vGPCR in LEC, and matrix metalloproteinases, which could act in concert with Ang2 to contribute to KS development. These findings support the paracrine and autocrine roles of the lytic KSHV-encoded proteins, vIL6 and vGPCR, in KS pathogenesis and identify Ang2 as a potential therapeutic target for this neoplasm.

Low molecular weight heparin and direct oral anticoagulants influence tumour formation, growth, invasion and vascularisation by separate mechanisms.

The bidirectional association between coagulation and cancer has been established. However, anticoagulant therapies have been reported to have beneficial outcomes by influencing the vascularisation of the tumours. In this study the influence of a set of anticoagulants on tumour formation, invasion and vascularisation was examined. WM-266-4 melanoma and AsPC-1 pancreatic cancer cell lines were treated with LMWH (Tinzaparin and Dalteparin), and DOAC (Apixaban and Rivaroxaban) and the rate of tumour formation, growth and invasion were measured in vitro. In addition, the influence of these anticoagulants on vascularisation was examined using the chorioallantoic membrane assay (CAM) model and compared to the outcome of treatment with Bevacizumab. Using this model the influence of pharmacological concentrations of the anticoagulant on the growth, invasion and vascularisation of tumours derived from WM-266-4 and AsPC-1 cells was also measured in vivo. Tinzaparin and Daltepain reduced tumour formation and invasion by the cell lines in vitro, but with dissimilar potencies. In addition, treatment of CAM with LMWH reduced the local vascular density beyond that achievable with Bevacizumab, particularly suppressing the formation of larger-diameter blood vessels. In contrast, treatment with DOAC was largely ineffective. Treatment of CAM-implanted tumours with LMWH also reduced tumour vascularisation, while treatment of tumours with Apixaban reduced tumour growth in vivo. In conclusion, LMWH and DOAC appear to have anti-cancer properties that are exerted through different mechanisms.

Expression of terminally glycosylated calcitonin receptor-like receptor in uterine leiomyoma: endothelial phenotype and association with microvascular density.

PURPOSE: The role for the hypoxia-inducible angiogenic factor adrenomedullin (AM) in tumor growth and progression has been suggested. Calcitonin receptor-like receptor (CL) is a G protein-coupled receptor (GPCR) that mediates effects of AM, but little information is available on its expression and functional state in human tumors. The present study attempted to determine CL potential for antiangiogenic therapy of uterine leiomyoma. EXPERIMENTAL DESIGN AND RESULTS: GPCR CL is transported to the cell surface and recognized by AM only when terminally/mature glycosylated. The presence and localization of this form of the receptor in tumor and surrounding myometrial tissues obtained from leiomyoma-bearing uteri were examined using deglycosylation, immunoblotting, and immunofluorescence analysis. The mature CL glycoprotein was expressed in both tissues and localized exclusively in normal and tumor endothelium within leiomyoma-bearing uteri. The functionality of the receptor expressed in myometrial microvascular endothelial cells (MMVEC) was examined in vitro using receptor internalization and angiogenic assays. The mature CL glycoprotein expressed by primary MMVECs was functional because AM interacted with this GPCR and induced its internalization as well as angiogenic effects (proliferation and migration) in MMVECs in vitro. Finally, the levels of tissue-expressed mature CL glycoprotein as a functional form of this GPCR were analyzed by immunoblotting. The expression of this functional form of the receptor in vivo was significantly decreased (P = 0.01) in leiomyoma tissue, and this was concurrent with the decrease in microvascular density (measured by Chalkley counting) in tumor compared with surrounding myometrium (P = 0.031). CONCLUSIONS: Our findings suggest that GPCR CL mediates angiogenic effects of AM in myometrium and that further evaluation of the properties of the CL expressed in both normal and tumor endothelium in vivo may be essential before targeting this endothelial GPCR for antiangiogenic therapies.

Antiangiogenic activity of a domain deletion mutant of tissue plasminogen activator containing kringle 2.

OBJECTIVE: The thrombolytic therapy drug, Reteplase, is a domain deletion mutant of tissue plasminogen activator (tPA), comprising the kringle 2 and protease (K2P) domains. Some kringle domains of hemostatic proteins are antiangiogenic and promote apoptosis. The objective of this study was to investigate whether K2P is an angiogenesis inhibitor because of the presence of kringle 2. METHODS AND RESULTS: K2P inhibited basic fibroblast growth factor-induced human endothelial cell proliferation and migration. Inhibition was not dependent on the protease activity of K2P because similar results were obtained with catalytically inactivated K2P. Purification of the kringle 2 domain derived from elastase cleavage of K2P at the Arg275-Ile276 bond revealed that inhibition was mediated by this domain. In addition, K2P inhibited angiogenesis in vivo and increased endothelial cell apoptosis. CONCLUSIONS: Wound healing and angiogenesis are severely compromised by K2P. These data provide new mechanistic insights into the bleeding complications observed in some patients while undergoing thrombolytic therapy with this drug. In addition, we identify the kringle 2 domain of tPA as a novel target for antiangiogenic therapy.

Transcriptional regulation of the CRLR gene in human microvascular endothelial cells by hypoxia.

Adrenomedullin is a 52 amino acid peptide that shows a remarkable range of effects on the vasculature that include inter alia, vasodilatation, regulation of permeability, inhibition of endothelial cell apoptosis, and promotion of angiogenesis. Recently the G-protein coupled receptor (GPCR) calcitonin receptor-like receptor (CRLR), and receptor activity modifying proteins (RAMPs) have become recognized as integral components of the adrenomedullin signaling system. However, mechanisms of regulation of CRLR expression are still largely unknown. This is in part due to lack of information on the gene promoter. In this study we have determined the transcriptional start of human CRLR cDNA by 5'-RACE and cloned the proximal 5'-flanking region of the gene by PCR. The 2318 bp genomic fragment contains the basal promoter of human CRLR, including potential TATA-boxes and several GC boxes. Regulatory elements binding known transcription factors, such as Sp-1, Pit-1, glucocorticoid receptor, and hypoxia-inducible factor-1 alpha (HIF-1alpha) were also identified. When cloned into reporter gene vectors, the genomic fragment showed significant promoter activity, indicating that the 5'-flanking region isolated by PCR contains the gene promoter of human CRLR. Of significance is that the cloned promoter fragments were activated by hypoxia when transfected in primary microvascular endothelial cells. Site-directed mutagenesis of the consensus hypoxia-response element (HRE) in the 5'-flanking region abolished such a response. We also demonstrated by semi-quantitative RT-PCR that transcription of the gene is activated by hypoxia in microvascular endothelial cells. In contrast, expression of RAMPs 1, 2, and 3 was unaffected by low oxygen tension. We conclude that simultaneous transcriptional up-regulation of CRLR and its ligand adrenomedullin in endothelial cells could lead to a potent survival loop and therefore might play a significant role in vascular responses to hypoxia and ischemia.

The G-protein-coupled receptor CLR is upregulated in an autocrine loop with adrenomedullin in clear cell renal cell carcinoma and associated with poor prognosis.

PURPOSE: The G-protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) and its ligand peptide adrenomedullin (encoded by ADM gene) are implicated in tumor angiogenesis in mouse models but poorly defined in human cancers. We therefore investigated the diagnostic/prognostic use for CLR in human tumor types that may rely on adrenomedullin signaling and in clear cell renal cell carcinoma (RCC), a highly vascular tumor, in particular. EXPERIMENTAL DESIGN: In silico gene expression mRNA profiling microarray study (n = 168 tumors) and cancer profiling cDNA array hybridization (n = 241 pairs of patient-matched tumor/normal tissue samples) were carried out to analyze ADM mRNA expression in 13 tumor types. Immunohistochemistry on tissue microarrays containing patient-matched renal tumor/normal tissues (n = 87 pairs) was conducted to study CLR expression and its association with clinicopathologic parameters and disease outcome. RESULTS: ADM expression was significantly upregulated only in RCC and endometrial adenocarcinoma compared with normal tissue counterparts (P < 0.01). CLR was localized in tumor cells and vessels in RCC and upregulated as compared with patient-matched normal control kidney (P < 0.001). Higher CLR expression was found in advanced stages (P < 0.05), correlated with high tumor grade (P < 0.01) and conferred shorter overall survival (P < 0.01). CONCLUSIONS: In human tissues ADM expression is upregulated in cancer type-specific manner, implicating potential role for adrenomedullin signaling in particular in RCC, where CLR localization suggests autocrine/paracrine mode for adrenomedullin action within the tumor microenvironment. Our findings reveal previously unrecognized CLR upregulation in an autocrine loop with adrenomedullin in RCC with potential application for this GPCR as a target for future functional studies and drug development.

Adrenomedullin haploinsufficiency predisposes to secondary lymphedema.

Secondary lymphedema is a debilitating condition, and genetic factors predisposing to its development remain largely unknown. Adrenomedullin (AM) is peptide encoded, together with proadrenomedullin N-terminal peptide (PAMP), by the Adm gene (adrenomedullin gene). AM and its putative receptor calcitonin receptor-like receptor (CLR) are implicated in angiogenesis and lymphangiogenesis during embryogenesis and wound healing, suggesting their possible involvement in secondary lymphedema. To investigate whether AM deficiency predisposes to secondary lymphedema, we used heterozygous adult mice with Adm gene-knockin stop mutation, which selectively abrogated AM, but preserved PAMP, expression (Adm(AM+/Δ) animals). After hind limb skin incision, Adm messenger RNA expression was upregulated in wounded tissue of both Adm(AM+/+) and Adm(AM+/Δ) mice. However, only Adm(AM+/Δ) animals developed limb swelling and histopathological lymphedematous changes, including epidermal thickening, elevated collagen fiber density, and increased microvessel diameter. Secondary lymphedema was prevented when circulating AM levels in Adm(AM+/Δ) mice were restored by systemic peptide delivery. In human skin, CLR was expressed in tissue components affected by lymphedema, including epidermis, lymphatics, and blood vessels. Our study identified a previously unrecognized role for endogenous AM as a key factor in secondary lymphedema pathogenesis and provided experimental in vivo evidence of an underlying germ-line genetic predisposition to developing this disorder.

The expression and regulation of adrenomedullin in the human endometrium: a candidate for endometrial repair.

After menstruation, the endometrium has a remarkable capacity for repair, but the factors involved remain undefined. We hypothesize adrenomedullin (AM) plays a role in this process. Premenstrually progesterone levels decline, stimulating prostaglandin (PG) synthesis, vasoconstriction, and hypoxia. This study aimed to determine 1) AM expression throughout the menstrual (M) cycle and 2) its regulation by PG and hypoxia. Human endometrial biopsies (n = 51) were collected with ethical approval and consent. AM mRNA expression was examined by quantitative RT-PCR and was found to be selectively elevated in endometrium from the menstrual (M) phase (P < 0.001). AM immunohistochemical staining was maximal in M and proliferative (P) endometrium. Culture of secretory, but not P, explants with 100 nm PGF(2α) or hypoxia (0.5% O2) increased AM mRNA (P < 0.05). P explants were induced to increase AM expression using in vitro progesterone withdrawal but required the presence of hypoxia (P < 0.05). Short hairpin sequences against hypoxia-inducible factor-1α (HIF-1α) inhibited AM hypoxic up-regulation but did not alter PGF(2α)-induced expression. The AM receptor was immunolocalized to endothelial cells in both lymphatic and blood vessels. Conditioned medium from PGF(2α)-treated cells increased endothelial cell proliferation and branching (P < 0.05). This was abolished by AM receptor antagonists. In conclusion, AM is elevated at the time of endometrial repair and induces both angiogenesis and lymphangiogenesis by stimulating endothelial cell proliferation and tube formation. In the human endometrium, AM expression is up-regulated by two mechanisms: a HIF-1α-mediated hypoxic induction and a HIF-1α-independent PGF(2α) pathway. These physiological mechanisms may provide novel therapeutic targets for disorders such as heavy menstrual bleeding.

Adrenomedullin and CGRP interact with endogenous calcitonin-receptor-like receptor in endothelial cells and induce its desensitisation by different mechanisms.

Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) are related peptides with distinct pharmacological profiles. Calcitonin-receptor-like receptor (CRLR, now known as CL) can function as either an AM receptor or a CGRP receptor, when cotransfected with receptor-activity-modifying proteins (RAMPs) that define ligand-binding specificity. The aim of the present study was to determine the role of endogenously expressed CL (EndoCL) in generating endogenous AM and CGRP receptors. We raised anti-human CL antibody and identified microvascular endothelial cells (MVECs) as a major CL-expressing cell type in tissues by immunohistochemistry. Cultured MVECs continue to express EndoCL as well as fully active endogenous AM- and CGRP-sensitive receptors in vitro, as demonstrated by the ability of both peptides to induce migration and Akt phosphorylation. We therefore tested the hypothesis that endothelial EndoCL can interact with both AM and CGRP by examining receptor internalisation and desensitisation (loss of the ability to induce Akt phosphorylation). We found that agonist-mediated internalisation of EndoCL occurs in response to AM but not CGRP in MVECs. However, AM-induced EndoCL internalisation was blocked by antagonists of both AM and CGRP receptors: AM(22-52) and CGRP(8-37), respectively. Furthermore, AM-induced EndoCL internalisation resulted in desensitisation not only of AM but also of CGRP receptors. Finally, CGRP also induced desensitisation of both endogenous AM and CGRP receptors, but did not mediate EndoCL internalisation despite interaction with this receptor. Thus, EndoCL interacts with both AM and CGRP, and simultaneously acts as a receptor for both peptides (i.e acting as an endogenous AM/CGRP receptor) in endothelial cells. Interaction with either ligand is sufficient to induce EndoCL desensitisation to both AM and CGRP, but differential mechanisms are involved since only AM induces EndoCL internalisation. These novel findings regarding regulation of EndoCL function in endothelial cells are likely to be of importance in conditions where AM or CGRP levels are elevated, such as cardiovascular disease, diabetes and inflammation.

Adrenomedullin: multiple functions in human pregnancy.

Adrenomedullin is a 52 amino acid peptide originally isolated from human phaeochromocytoma in 1993. It was initially demonstrated to have profound effects on the vasculature including vasodilatation and subsequently promotion of angiogenesis. Since then it has become apparent that it has a wide range of other biological actions including regulation of cell growth and differentiation. Successful pregnancy outcome relies on establishing and maintaining throughout gestation an efficient blood supply to the fetus. This allows the exchange of nutrients, oxygenation of fetal blood and removal of cytotoxins from the fetus, such as carbon dioxide. One of the most important local adaptations to pregnancy is the change in maternal blood flow to the implantation site. Evidence now points towards a vital role for adrenomedullin in the regulation of placentation. It appears that adrenomedullin may play important roles in the regulation of fetal perfusion both in normal and in compromised pregnancies. However, most studies have focused on measuring adrenomedullin levels and studying its expression as well as that of its receptors. More functional studies are now required to elucidate the underlying mechanisms involved.