Cancer Burden Is Controlled by Mural Cell-ß3-Integrin Regulated Crosstalk with Tumor Cells.

Enhanced blood vessel (BV) formation is thought to drive tumor growth through elevated nutrient delivery. However, this observation has overlooked potential roles for mural cells in directly affecting tumor growth independent of BV function. Here we provide clinical data correlating high percentages of mural-ß3-integrin-negative tumor BVs with increased tumor sizes but no effect on BV numbers. Mural-ß3-integrin loss also enhances tumor growth in implanted and autochthonous mouse tumor models with no detectable effects on BV numbers or function. At a molecular level, mural-cell ß3-integrin loss enhances signaling via FAK-p-HGFR-p-Akt-p-p65, driving CXCL1, CCL2, and TIMP-1 production. In particular, mural-cell-derived CCL2 stimulates tumor cell MEK1-ERK1/2-ROCK2-dependent signaling and enhances tumor cell survival and tumor growth. Overall, our data indicate that mural cells can control tumor growth via paracrine signals regulated by ß3-integrin, providing a previously unrecognized mechanism of cancer growth control.

ERG activity is regulated by endothelial FAK coupling with TRIM25/USP9x in vascular patterning.

Precise vascular patterning is crucial for normal growth and development. The ERG transcription factor drives Delta-like ligand 4 (DLL4)/Notch signalling and is thought to act as a pivotal regulator of endothelial cell (EC) dynamics and developmental angiogenesis. However, molecular regulation of ERG activity remains obscure. Using a series of EC-specific focal adhesion kinase (FAK)-knockout (KO) and point-mutant FAK-knock-in mice, we show that loss of ECFAK, its kinase activity or phosphorylation at FAK-Y397, but not FAK-Y861, reduces ERG and DLL4 expression levels together with concomitant aberrations in vascular patterning. Rapid immunoprecipitation mass spectrometry of endogenous proteins identified that endothelial nuclear-FAK interacts with the deubiquitinase USP9x and the ubiquitin ligase TRIM25. Further in silico analysis confirms that ERG interacts with USP9x and TRIM25. Moreover, ERG levels are reduced in FAKKO ECs via a ubiquitin-mediated post-translational modification programme involving USP9x and TRIM25. Re-expression of ERG in vivo and in vitro rescues the aberrant vessel-sprouting defects observed in the absence of ECFAK. Our findings identify ECFAK as a regulator of retinal vascular patterning by controlling ERG protein degradation via TRIM25/USP9x.

Elucidating the role of the kinase activity of endothelial cell focal adhesion kinase in angiocrine signalling and tumour growth.

A common limitation of cancer treatments is chemotherapy resistance. We have previously identified that endothelial cell (EC)-specific deletion of focal adhesion kinase (FAK) sensitises tumour cells to DNA-damaging therapies, reducing tumour growth in mice. The present study addressed the kinase activity dependency of EC FAK sensitisation to the DNA-damaging chemotherapeutic drug, doxorubicin. FAK is recognised as a therapeutic target in tumour cells, leading to the development of a range of inhibitors, the majority being ATP competitive kinase inhibitors. We demonstrate that inactivation of EC FAK kinase domain (kinase dead; EC FAK-KD) in established subcutaneous B16F0 tumours improves melanoma cell sensitisation to doxorubicin. Doxorubicin treatment in EC FAK-KD mice reduced the percentage change in exponential B16F0 tumour growth further than in wild-type mice. There was no difference in tumour blood vessel numbers, vessel perfusion or doxorubicin delivery between genotypes, suggesting a possible angiocrine effect on the regulation of tumour growth. Doxorubicin reduced perivascular malignant cell proliferation, while enhancing perivascular tumour cell apoptosis and DNA damage in tumours grown in EC FAK-KD mice 48 h after doxorubicin injection. Human pulmonary microvascular ECs treated with the pharmacological FAK kinase inhibitors defactinib, PF-562,271 or PF-573,228 in combination with doxorubicin also reduced cytokine expression levels. Together, these data suggest that targeting EC FAK kinase activity may alter angiocrine signals that correlate with improved acute tumour cell chemosensitisation. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Phosphorylation of pericyte FAK-Y861 affects tumour cell apoptosis and tumour blood vessel regression.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is overexpressed in many cancer types and in vivo studies have shown that vascular endothelial cell FAK expression and FAK-phosphorylation at tyrosine (Y) 397, and subsequently FAK-Y861, are important in tumour angiogenesis. Pericytes also play a vital role in regulating tumour blood vessel stabilisation, but the specific involvement of pericyte FAK-Y397 and FAK-Y861 phosphorylation in tumour blood vessels is unknown. Using PdgfrßCre + ;FAKWT/WT, PdgfrßCre + ;FAKY397F/Y397F and PdgfrßCre + ;FAKY861F/Y861F mice, our data demonstrate that Lewis lung carcinoma tumour growth, tumour blood vessel density, blood vessel perfusion and pericyte coverage were affected only in late stage tumours in PdgfrßCre + ;FAKY861F/Y861F but not PdgfrßCre + ;FAKY397F/Y397F mice. Further examination indicates a dual role for pericyte FAK-Y861 phosphorylation in the regulation of tumour vessel regression and also in the control of pericyte derived signals that influence apoptosis in cancer cells. Overall this study identifies the role of pericyte FAK-Y861 in the regulation of tumour vessel regression and tumour growth control and that non-phosphorylatable FAK-Y861F in pericytes reduces tumour growth and blood vessel density.

C-type natriuretic peptide co-ordinates cardiac structure and function.

AIMS: C-type natriuretic peptide (CNP) is an essential endothelium-derived signalling species that governs vascular homoeostasis; CNP is also expressed in the heart but an intrinsic role for the peptide in cardiac function is not established. Herein, we employ unique transgenic strains with cell-specific deletion of CNP to define a central (patho)physiological capacity of CNP in maintaining heart morphology and contractility. METHODS AND RESULTS: Cardiac structure and function were explored in wild type (WT), cardiomyocyte (cmCNP-/-), endothelium (ecCNP-/-), and fibroblast (fbCNP-/-)-specific CNP knockout mice, and global natriuretic peptide receptor (NPR)-B-/-, and NPR-C-/- animals at baseline and in experimental models of myocardial infarction and heart failure (HF). Endothelium-specific deletion of CNP resulted in impaired coronary responsiveness to endothelium-dependent- and flow-mediated-dilatation; changes mirrored in NPR-C-/- mice. Ex vivo, global ischaemia resulted in larger infarcts and diminished functional recovery in cmCNP-/- and NPR-C-/-, but not ecCNP-/-, vs. WT. The cardiac phenotype of cmCNP-/-, fbCNP-/-, and NPR-C-/- (but not ecCNP-/- or NPR-B-/-) mice was more severe in pressure overload- and sympathetic hyperactivation-induced HF compared with WT; these adverse effects were rescued by pharmacological CNP administration in WT, but not NPR-C-/-, mice. At a molecular level, CNP/NPR-C signalling is impaired in human HF but attenuates activation of well-validated pro-hypertrophic and pro-fibrotic pathways. CONCLUSION: C-type natriuretic peptide of cardiomyocyte, endothelial and fibroblast origins co-ordinates and preserves cardiac structure, function, and coronary vasoreactivity via activation of NPR-C. Targeting NPR-C may prove an innovative approach to treating HF and ischaemic cardiovascular disorders.

Repurposing an anti-cancer agent for the treatment of hypertrophic heart disease.

Coronary microvascular dysfunction combined with maladaptive cardiomyocyte morphology and energetics is a major contributor to heart failure advancement. Thus, dually enhancing cardiac angiogenesis and targeting cardiomyocyte function to slow, or reverse, the development of heart failure is a logical step towards improved therapy. We present evidence for the potential to repurpose a former anti-cancer Arg-Gly-Asp (RGD)-mimetic pentapeptide, cilengitide, here used at low doses. Cilengitide targets αvß3 integrin and this protein is upregulated in human dilated and ischaemic cardiomyopathies. Treatment of mice after abdominal aortic constriction (AAC) surgery with low-dose cilengitide (ldCil) enhances coronary angiogenesis and directly affects cardiomyocyte hypertrophy with an associated reduction in disease severity. At a molecular level, ldCil treatment has a direct effect on cardiac endothelial cell transcriptomic profiles, with a significant enhancement of pro-angiogenic signalling pathways, corroborating the enhanced angiogenic phenotype after ldCil treatment. Moreover, ldCil treatment of Angiotensin II-stimulated AngII-stimulated cardiomyocytes significantly restores transcriptomic profiles similar to those found in normal human heart. The significance of this finding is enhanced by transcriptional similarities between AngII-treated cardiomyocytes and failing human hearts. Taken together, our data provide evidence supporting a possible new strategy for improved heart failure treatment using low-dose RGD-mimetics with relevance to human disease. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Endothelial-cell FAK targeting sensitizes tumours to DNA-damaging therapy.

Chemoresistance is a serious limitation of cancer treatment. Until recently, almost all the work done to study this limitation has been restricted to tumour cells. Here we identify a novel molecular mechanism by which endothelial cells regulate chemosensitivity. We establish that specific targeting of focal adhesion kinase (FAK; also known as PTK2) in endothelial cells is sufficient to induce tumour-cell sensitization to DNA-damaging therapies and thus inhibit tumour growth in mice. The clinical relevance of this work is supported by our observations that low blood vessel FAK expression is associated with complete remission in human lymphoma. Our study shows that deletion of FAK in endothelial cells has no apparent effect on blood vessel function per se, but induces increased apoptosis and decreased proliferation within perivascular tumour-cell compartments of doxorubicin- and radiotherapy-treated mice. Mechanistically, we demonstrate that endothelial-cell FAK is required for DNA-damage-induced NF-κB activation in vivo and in vitro, and the production of cytokines from endothelial cells. Moreover, loss of endothelial-cell FAK reduces DNA-damage-induced cytokine production, thus enhancing chemosensitization of tumour cells to DNA-damaging therapies in vitro and in vivo. Overall, our data identify endothelial-cell FAK as a regulator of tumour chemosensitivity. Furthermore, we anticipate that this proof-of-principle data will be a starting point for the development of new possible strategies to regulate chemosensitization by targeting endothelial-cell FAK specifically.

Single-cell transcriptome analyses reveal novel targets modulating cardiac neovascularization by resident endothelial cells following myocardial infarction.

AIMS: A better understanding of the pathways that regulate regeneration of the coronary vasculature is of fundamental importance for the advancement of strategies to treat patients with heart disease. Here, we aimed to investigate the origin and clonal dynamics of endothelial cells (ECs) associated with neovascularization in the adult mouse heart following myocardial infarction (MI). Furthermore, we sought to define murine cardiac endothelial heterogeneity and to characterize the transcriptional profiles of pro-angiogenic resident ECs in the adult mouse heart, at single-cell resolution. METHODS AND RESULTS: An EC-specific multispectral lineage-tracing mouse (Pdgfb-iCreERT2-R26R-Brainbow2.1) was used to demonstrate that structural integrity of adult cardiac endothelium following MI was maintained through clonal proliferation by resident ECs in the infarct border region, without significant contributions from bone marrow cells or endothelial-to-mesenchymal transition. Ten transcriptionally discrete heterogeneous EC states, as well as the pathways through which each endothelial state is likely to enhance neovasculogenesis and tissue regeneration following ischaemic injury were defined. Plasmalemma vesicle-associated protein (Plvap) was selected for further study, which showed an endothelial-specific and increased expression in both the ischaemic mouse and human heart, and played a direct role in regulating human endothelial proliferation in vitro. CONCLUSION: We present a single-cell gene expression atlas of cardiac specific resident ECs, and the transcriptional hierarchy underpinning endogenous vascular repair following MI. These data provide a rich resource that could assist in the development of new therapeutic interventions to augment endogenous myocardial perfusion and enhance regeneration in the injured heart.

Dual role of pericyte α6ß1-integrin in tumour blood vessels.

The α6ß1-integrin is a major laminin receptor, and formation of a laminin-rich basement membrane is a key feature in tumour blood vessel stabilisation and pericyte recruitment, processes that are important in the growth and maturation of tumour blood vessels. However, the role of pericyte α6ß1-integrin in angiogenesis is largely unknown. We developed mice where the α6-integrin subunit is deleted in pericytes and examined tumour angiogenesis and growth. These mice had: (1) reduced pericyte coverage of tumour blood vessels; (2) reduced tumour blood vessel stability; (3) increased blood vessel diameter; (4) enhanced blood vessel leakiness, and (5) abnormal blood vessel basement membrane architecture. Surprisingly, tumour growth, blood vessel density and metastasis were not altered. Analysis of retinas revealed that deletion of pericyte α6-integrin did not affect physiological angiogenesis. At the molecular level, we provide evidence that pericyte α6-integrin controls PDGFRß expression and AKT-mTOR signalling. Taken together, we show that pericyte α6ß1-integrin regulates tumour blood vessels by both controlling PDGFRß and basement membrane architecture. These data establish a novel dual role for pericyte α6-integrin as modulating the blood vessel phenotype during pathological angiogenesis.

Focal Adhesion Kinase (FAK) tyrosine 397E mutation restores the vascular leakage defect in endothelium-specific FAK-kinase dead mice.

Focal adhesion kinase (FAK) inhibitors have been developed as potential anticancer agents and are undergoing clinical trials. In vitro activation of the FAK kinase domain triggers autophosphorylation of Y397, Src activation, and subsequent phosphorylation of other FAK tyrosine residues. However, how FAK Y397 mutations affect FAK kinase-dead (KD) phenotypes in tumour angiogenesis in vivo is unknown. We developed three Pdgfb-iCreert -driven endothelial cell (EC)-specific, tamoxifen-inducible homozygous mutant mouse lines: FAK wild-type (WT), FAK KD, and FAK double mutant (DM), i.e. KD with a putatively phosphomimetic Y397E mutation. These ECCre+;FAKWT/WT , ECCre+;FAKKD/KD and ECCre+;FAKDM/DM mice were injected subcutaneously with syngeneic B16F0 melanoma cells. Tumour growth and tumour blood vessel functions were unchanged between ECCre+;FAKWT/WT and ECCre-;FAKWT/WT control mice. In contrast, tumour growth and vessel density were decreased in ECCre+;FAKKD/KD and ECCre+;FAKDM/DM mice, as compared with Cre - littermates. Despite no change in the percentage of perfused vessels or pericyte coverage in either genotype, tumour hypoxia was elevated in ECCre+;FAKKD/KD and ECCre+;FAKDM/DM mice. Furthermore, although ECCre+;FAKKD/KD mice showed reduced blood vessel leakage, ECCre+;FAKDM/DM and ECCre-;FAKDM/DM mice showed no difference in leakage. Mechanistically, fibronectin-stimulated Y397 autophosphorylation was reduced in Cre+;FAKKD/KD ECs as compared with Cre+;FAKWT/WT cells, with no change in phosphorylation of the known Src targets FAK-Y577, FAK-Y861, FAK-Y925, paxillin-Y118, p130Cas-Y410. Cre+;FAKDM/DM ECs showed decreased Src target phosphorylation levels, suggesting that the Y397E substitution actually disrupted Src activation. Reduced VE-cadherin-pY658 levels in Cre+;FAKKD/KD ECs were rescued in Cre+FAKDM/DM ECs, corresponding with the rescue in vessel leakage in the ECCre+;FAKDM/DM mice. We show that EC-specific FAK kinase activity is required for tumour growth, angiogenesis, and vascular permeability. The ECCre+;FAKDM/DM mice restored the KD-dependent tumour vascular leakage observed in ECCre+;FAKKD/KD mice in vivo. This study opens new fields in in vivo FAK signalling. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Overcoming the Lack of Oral Availability of Cyclic Hexapeptides: Design of a Selective and Orally Available Ligand for the Integrin†αvß3.

A highly systematic approach for the development of both orally bioavailable and bioactive cyclic N-methylated hexapeptides as high affinity ligands for the integrin αvß3 is based on two concepts: a) screening of systematically designed libraries with spatial diversity and b) masking of the peptide charge with a lipophilic protecting group. The key steps of the method are 1) initial design of a combinatorial library of N-methylated analogues of the stem peptide cyclo(d-Ala-Ala5 ); 2) selection of cyclic peptides with the highest intestinal permeability; 3) design of sublibraries with the bioactive RGD sequence in all possible positions; 4) selection of the best ligands for RGD-recognizing integrin subtypes; 5) fine-tuning of the affinity and selectivity by additional Ala to Xaa substitutions; 6) protection of the charged functional groups according to the prodrug concept to regain intestinal and oral permeability; 7) proof of biological effects in mice after oral administration.

Pancreatic stellate cells regulate blood vessel density in the stroma of pancreatic ductal adenocarcinoma.

BACKGROUND/OBJECTIVES: The vascular heterogeneity of pancreatic ductal adenocarcinoma (PDAC) has never been characterised. We analysed the heterogeneous vascular density of human PDAC along with its prognostic correlation. METHODS: Tissue Microarrays of 87 patients with different pancreatico-biliary pathologies were analysed in an automated manner (Ariol™) after CD31 staining to assess vascular density in juxta-tumoral and panstromal compartments. In vitro and ex vivo assays were carried out to assess the role of PSC. RESULTS: PDAC has a distinct vascular density and distribution of vessels compared to cholangiocarcinoma. The PDAC juxta-tumoral stroma was hypovascular and the normal adjacent rim was hypervascular compared to the panstromal compartment. These features adversely affected patient prognosis, suggesting a model for spatio-temporal PDAC evolution. Mice aortic rings and 3D organotypic cultures demonstrated pro- and anti-angiogenic signalling from activated PSC and cancer cells respectively. ATRA-induced quiescence suppressed the pro-angiogenic activity of PSC. CONCLUSION: Human PDAC has variable vascularity at microscopic level suggesting that novel stromal directed therapies would need to be determined by pathological characteristics.

Acute depletion of endothelial ß3-integrin transiently inhibits tumor growth and angiogenesis in mice.

RATIONALE: The dramatic upregulation of αvß3-integrin that occurs in the vasculature during tumor growth has long suggested that the endothelial expression of this molecule is an ideal target for antiangiogenic therapy to treat cancer. This discovery led to the development of small-molecule inhibitors directed against αvß3-integrin that are currently in clinical trials. In 2002, we reported that ß3-integrin-knockout mice exhibit enhanced tumor growth and angiogenesis. However, as ß3-integrin is expressed by a wide variety of cells, endothelial cell-specific contributions to tumor angiogenesis are muddied by the use of a global knockout of ß3-integrin function. OBJECTIVE: Our aim was to examine the endothelial-specific contribution ß3-integrin makes to tumor growth and angiogenesis. METHODS AND RESULTS: We have crossed ß3-integrin-floxed (ß3-floxed) mice to 2 endothelial-specific Cre models and examined angiogenic responses in vivo, ex vivo, and in vitro. We show that acute depletion of endothelial ß3-integrin inhibits tumor growth and angiogenesis preventatively, but not in already established tumors. However, the effects are transient, and long-term depletion of the molecule is ineffective. Furthermore, long-term depletion of the molecule correlates with many molecular changes, such as reduced levels of focal adhesion kinase expression and a misbalance in focal adhesion kinase phosphorylation, which may lead to a release from the inhibitory effects of decreased endothelial ß3-integrin expression. CONCLUSIONS: Our findings imply that timing and length of inhibition are critical factors that need to be considered when targeting the endothelial expression of ß3-integrin to inhibit tumor growth and angiogenesis.

Tumour angiogenesis is reduced in the Tc1 mouse model of Down's syndrome.

Down's syndrome (DS) is a genetic disorder caused by full or partial trisomy of human chromosome 21 and presents with many clinical phenotypes including a reduced incidence of solid tumours. Recent work with the Ts65Dn model of DS, which has orthologues of about 50% of the genes on chromosome 21 (Hsa21), has indicated that three copies of the ETS2 (ref. 3) or DS candidate region 1 (DSCR1) genes (a previously known suppressor of angiogenesis) is sufficient to inhibit tumour growth. Here we use the Tc1 transchromosomic mouse model of DS to dissect the contribution of extra copies of genes on Hsa21 to tumour angiogenesis. This mouse expresses roughly 81% of Hsa21 genes but not the human DSCR1 region. We transplanted B16F0 and Lewis lung carcinoma tumour cells into Tc1 mice and showed that growth of these tumours was substantially reduced compared with wild-type littermate controls. Furthermore, tumour angiogenesis was significantly repressed in Tc1 mice. In particular, in vitro and in vivo angiogenic responses to vascular endothelial growth factor (VEGF) were inhibited. Examination of the genes on the segment of Hsa21 in Tc1 mice identified putative anti-angiogenic genes (ADAMTS1and ERG) and novel endothelial cell-specific genes, never previously shown to be involved in angiogenesis (JAM-B and PTTG1IP), that, when overexpressed, are responsible for inhibiting angiogenic responses to VEGF. Three copies of these genes within the stromal compartment reduced tumour angiogenesis, explaining the reduced tumour growth in DS. Furthermore, we expect that, in addition to the candidate genes that we show to be involved in the repression of angiogenesis, the Tc1 mouse model of DS will permit the identification of other endothelium-specific anti-angiogenic targets relevant to a broad spectrum of cancer patients.

alphavbeta3 integrin and angiogenesis: a moody integrin in a changing environment.

In the adult, angiogenesis, the formation of new blood vessels from pre-existing vasculature contributes to the pathogenesis of many disorders including cancer. The role of adhesion molecules, especially integrins, in pathological angiogenesis has long been the subject of investigation, mostly because of their potential as anti-angiogenic targets. Recent studies have highlighted the complexities connected with understanding the roles of one particular integrin, alphavbeta3, in neovascularization. This integrin is notoriously promiscuous and its precise functions in angiogenesis are unclear. Here, I have firstly summarized some of the salient features of the roles played by alphavbeta3 during angiogenesis; secondly attempted to address the apparently conflicting issues surrounding this topic; and finally raised some questions that appear to be unanswered.

Tumour-associated endothelial-FAK correlated with molecular sub-type and prognostic factors in invasive breast cancer.

BACKGROUND: Breast cancer is a heterogeneous disease that can be classified into one of 4 main molecular sub-types: luminal A, luminal B, Her2 over-expressing and basal-like (BL). These tumour sub-types require different treatments and have different risks of disease progression. BL cancers can be considered a sub-group of Triple negative (TN) cancers since they lack estrogen (ER), progesterone (PR) and Her2 expression. No targeted treatment currently exists for TN/BL cancers. Thus it is important to identify potential therapeutic targets and describe their relationship with established prognostic factors. Focal adhesion kinase (FAK) is upregulated in several human cancers and also plays a functional role in tumour angiogenesis. However, the association between breast cancer sub-types and tumour endothelial-FAK expression is unknown. METHODS: Using immunofluorescence, we quantified FAK expression in tumour endothelial and tumour cell compartments in 149 invasive breast carcinomas and correlated expression with clinical, pathological and molecular parameters. RESULTS: Low endothelial-FAK expression was independently associated with luminal A tumours at univariate (p < 0.001) and multivariate (p = 0.001) analysis. There was a positive correlation between FAK expression in the vascular and tumour cell compartments (Spearman's correlation co-efficient = 0.394, p < 0.001). Additionally, endothelial and tumour cell FAK expression were significantly increased in TN tumours (p = 0.043 and p = 0.033 respectively), in tumours with negative ER and PR status, and in high grade tumours at univariate analysis. CONCLUSION: Our findings establish a relationship between endothelial-FAK expression levels and the molecular sub-type of invasive breast cancer, and suggest that endothelial-FAK expression is potentially more clinically relevant than tumour cell FAK expression in breast cancer.

Treatment with αvß3-integrin-specific 29P attenuates pressure-overload induced cardiac remodelling after transverse aortic constriction in mice.

Heart failure remains one of the largest clinical burdens globally, with little to no improvement in the development of disease-eradicating therapeutics. Integrin targeting has been used in the treatment of ocular disease and cancer, but little is known about its utility in the treatment of heart failure. Here we sought to determine whether the second generation orally available, αvß3-specific RGD-mimetic, 29P , was cardioprotective. Male mice were subjected to transverse aortic constriction (TAC) and treated with 50 µg/kg 29P or volume-matched saline as Vehicle control. At 3 weeks post-TAC, echocardiography showed that 29P treatment significantly restored cardiac function and structure indicating the protective effect of 29P treatment in this model of heart failure. Importantly, 29P treatment improved cardiac function giving improved fractional shortening, ejection fraction, heart weight and lung weight to tibia length fractions, together with partial restoration of Ace and Mme levels, as markers of the TAC insult. At a tissue level, 29P reduced cardiomyocyte hypertrophy and interstitial fibrosis, both of which are major clinical features of heart failure. RNA sequencing identified that, mechanistically, this occurred with concomitant alterations to genes involved molecular pathways associated with these processes such as metabolism, hypertrophy and basement membrane formation. Overall, targeting αvß3 with 29P provides a novel strategy to attenuate pressure-overload induced cardiac hypertrophy and fibrosis, providing a possible new approach to heart failure treatment.

Contribution of ADAMTS1 as a tumor suppressor gene in human breast carcinoma. Linking its tumor inhibitory properties to its proteolytic activity on nidogen-1 and nidogen-2.

The extracellular protease ADAMTS1 (A disintegrin and metalloprotease with thrombospondin repeats 1) has been described as an anti-angiogenic molecule and its role as a putative tumor protective molecule has also been suggested. Here, we have used a tumor xenograft model to determine the role of ADAMTS1 in tumor growth and angiogenesis. Increasing levels of the protease led to the complete inhibition of tumor growth. In an attempt to elucidate the mechanism of action of this protease, we focused our attention on its proteolytic activity on nidogens, one of the main components of the vascular basement membrane. The increased expression of ADAMTS1 was accompanied by increased proteolysis of nidogen-1 and -2 and their almost complete removal from vascular structures, together with major morphological alterations of tumor blood vessels and a decreased vessel density. The clinical relevance of this work is supported by our observations that ADAMTS1 expression is decreased in breast tumor specimens when compared with healthy tissue. Our studies also reveal that the cleavage of nidogen-1 and -2 is partially inhibited in human tumor samples. Moreover, the deposition of both nidogens surrounding vascular structures is drastically altered, implying a possible reduction in the maintenance of vessel integrity. Our studies reflect the requirement to explore the functional interactions between proteases and specific substrates in cancer biology.

Focal adhesion kinase and tumour angiogenesis.

Angiogenesis, the formation of new blood vessels from pre-existing ones, is essential for tumour development. It is initiated and regulated by growth factors via their surface receptors, which activate several intracellular signalling pathways in endothelial cells. Cell adhesion molecules, such as integrins, also regulate angiogenesis. Despite these facts, inhibitors of endothelial cell growth factor receptors or integrins have not been as effective as initially hoped in the long-term inhibition of angiogenesis in cancer patients. Signalling downstream of growth factor receptors and integrins converge on the ubiquitously expressed non-receptor tyrosine kinase focal adhesion kinase (FAK). FAK is involved in endothelial cell proliferation, migration and survival, is up-regulated in many cancers and has recently been shown to control tumour angiogenesis. Indeed, FAK inhibitors are presently being developed for the treatment of cancer. However, recent studies have indicated the complexities of understanding the precise role for FAK in angiogenesis. Here we have summarized some of the key features of FAK, addressed some of the apparently contradictory roles of this molecule in angiogenesis and provided some perspectives for future studies.