Contribution of VEGF-B-Induced Endocardial Endothelial Cell Lineage in Physiological Versus Pathological Cardiac Hypertrophy.

BACKGROUND: Preclinical studies have shown the therapeutic potential of VEGF-B (vascular endothelial growth factor B) in revascularization of the ischemic myocardium, but the associated cardiac hypertrophy and adverse side effects remain a concern. To understand the importance of endothelial proliferation and migration for the beneficial versus adverse effects of VEGF-B in the heart, we explored the cardiac effects of autocrine versus paracrine VEGF-B expression in transgenic and gene-transduced mice. METHODS: We used single-cell RNA sequencing to compare cardiac endothelial gene expression in VEGF-B transgenic mouse models. Lineage tracing was used to identify the origin of a VEGF-B-induced novel endothelial cell population and adeno-associated virus-mediated gene delivery to compare the effects of VEGF-B isoforms. Cardiac function was investigated using echocardiography, magnetic resonance imaging, and micro-computed tomography. RESULTS: Unlike in physiological cardiac hypertrophy driven by a cardiomyocyte-specific VEGF-B transgene (myosin heavy chain alpha-VEGF-B), autocrine VEGF-B expression in cardiac endothelium (aP2 [adipocyte protein 2]-VEGF-B) was associated with septal defects and failure to increase perfused subendocardial capillaries postnatally. Paracrine VEGF-B led to robust proliferation and myocardial migration of a novel cardiac endothelial cell lineage (VEGF-B-induced endothelial cells) of endocardial origin, whereas autocrine VEGF-B increased proliferation of VEGF-B-induced endothelial cells but failed to promote their migration and efficient contribution to myocardial capillaries. The surviving aP2-VEGF-B offspring showed an altered ratio of secreted VEGF-B isoforms and developed massive pathological cardiac hypertrophy with a distinct cardiac vessel pattern. In the normal heart, we found a small VEGF-B-induced endothelial cell population that was only minimally expanded during myocardial infarction but not during physiological cardiac hypertrophy associated with mouse pregnancy. CONCLUSIONS: Paracrine and autocrine secretions of VEGF-B induce expansion of a specific endocardium-derived endothelial cell population with distinct angiogenic markers. However, autocrine VEGF-B signaling fails to promote VEGF-B-induced endothelial cell migration and contribution to myocardial capillaries, predisposing to septal defects and inducing a mismatch between angiogenesis and myocardial growth, which results in pathological cardiac hypertrophy.

PHD2 deletion in endothelial or arterial smooth muscle cells reveals vascular cell type-specific responses in pulmonary hypertension and fibrosis.

Hypoxia plays an important regulatory role in the vasculature to adjust blood flow to meet metabolic requirements. At the level of gene transcription, the responses are mediated by hypoxia-inducible factor (HIF) the stability of which is controlled by the HIF prolyl 4-hydroxylase-2 (PHD2). In the lungs hypoxia results in vasoconstriction, however, the pathophysiological relevance of PHD2 in the major arterial cell types; endothelial cells (ECs) and arterial smooth muscle cells (aSMCs) in the adult vasculature is incompletely characterized. Here, we investigated PHD2-dependent vascular homeostasis utilizing inducible deletions of PHD2 either in ECs (Phd2∆ECi) or in aSMCs (Phd2∆aSMC). Cardiovascular function and lung pathologies were studied using echocardiography, Doppler ultrasonography, intraventricular pressure measurement, histological, ultrastructural, and transcriptional methods. Cell intrinsic responses were investigated in hypoxia and in conditions mimicking hypertension-induced hemodynamic stress. Phd2∆ECi resulted in progressive pulmonary disease characterized by a thickened respiratory basement membrane (BM), alveolar fibrosis, increased pulmonary artery pressure, and adaptive hypertrophy of the right ventricle (RV). A low oxygen environment resulted in alterations in cultured ECs similar to those in Phd2∆ECi mice, involving BM components and vascular tone regulators favoring the contraction of SMCs. In contrast, Phd2∆aSMC resulted in elevated RV pressure without alterations in vascular tone regulators. Mechanistically, PHD2 inhibition in aSMCs involved  actin polymerization -related tension development via activated cofilin. The results also indicated that hemodynamic stress, rather than PHD2-dependent hypoxia response alone, potentiates structural remodeling of the extracellular matrix in the pulmonary microvasculature and respiratory failure.

Tyrosine-protein kinase Yes controls endothelial junctional plasticity and barrier integrity by regulating VE-cadherin phosphorylation and endocytosis.

Vascular endothelial (VE)-cadherin in endothelial adherens junctions is an essential component of the vascular barrier, critical for tissue homeostasis and implicated in diseases such as cancer and retinopathies. Inhibitors of Src cytoplasmic tyrosine kinase have been applied to suppress VE-cadherin tyrosine phosphorylation and prevent excessive leakage, edema and high interstitial pressure. Here we show that the Src-related Yes tyrosine kinase, rather than Src, is localized at endothelial cell (EC) junctions where it becomes activated in a flow-dependent manner. EC-specific Yes1 deletion suppresses VE-cadherin phosphorylation and arrests VE-cadherin at EC junctions. This is accompanied by loss of EC collective migration and exaggerated agonist-induced macromolecular leakage. Overexpression of Yes1 causes ectopic VE-cadherin phosphorylation, while vascular leakage is unaffected. In contrast, in EC-specific Src-deficiency, VE-cadherin internalization is maintained, and leakage is suppressed. In conclusion, Yes-mediated phosphorylation regulates constitutive VE-cadherin turnover, thereby maintaining endothelial junction plasticity and vascular integrity.

Computer extracted gland features from H&E predicts prostate cancer recurrence comparably to a genomic companion diagnostic test: a large multi-site study.

Existing tools for post-radical prostatectomy (RP) prostate cancer biochemical recurrence (BCR) prognosis rely on human pathologist-derived parameters such as tumor grade, with the resulting inter-reviewer variability. Genomic companion diagnostic tests such as Decipher tend to be tissue destructive, expensive, and not routinely available in most centers. We present a tissue non-destructive method for automated BCR prognosis, termed "Histotyping", that employs computational image analysis of morphologic patterns of prostate tissue from a single, routinely acquired hematoxylin and eosin slide. Patients from two institutions (n = 214) were used to train Histotyping for identifying high-risk patients based on six features of glandular morphology extracted from RP specimens. Histotyping was validated for post-RP BCR prognosis on a separate set of n = 675 patients from five institutions and compared against Decipher on n = 167 patients. Histotyping was prognostic of BCR in the validation set (p < 0.001, univariable hazard ratio [HR] = 2.83, 95% confidence interval [CI]: 2.03-3.93, concordance index [c-index] = 0.68, median years-to-BCR: 1.7). Histotyping was also prognostic in clinically stratified subsets, such as patients with Gleason grade group 3 (HR = 4.09) and negative surgical margins (HR = 3.26). Histotyping was prognostic independent of grade group, margin status, pathological stage, and preoperative prostate-specific antigen (PSA) (multivariable p < 0.001, HR = 2.09, 95% CI: 1.40-3.10, n = 648). The combination of Histotyping, grade group, and preoperative PSA outperformed Decipher (c-index = 0.75 vs. 0.70, n = 167). These results suggest that a prognostic classifier for prostate cancer based on digital images could serve as an alternative or complement to molecular-based companion diagnostic tests.

The Amino-Terminal Oligomerization Domain of Angiopoietin-2 Affects Vascular Remodeling, Mammary Gland Tumor Growth, and Lung Metastasis in Mice.

Angiopoietin-2 (ANGPT2) is a context-dependent TIE2 agonistic or antagonistic ligand that induces diverse responses in cancer. Blocking ANGPT2 provides a promising strategy for inhibiting tumor growth and metastasis, yet variable effects of targeting ANGPT2 have complicated drug development. ANGPT2443 is a naturally occurring, lower oligomeric protein isoform whose expression is increased in cancer. Here, we use a knock-in mouse line (mice expressing Angpt2443), a genetic model for breast cancer and metastasis (MMTV-PyMT), a syngeneic melanoma lung colonization model (B16F10), and orthotopic injection of E0771 breast cancer cells to show that alternative forms increase the diversity of Angpt2 function. In a mouse retina model of angiogenesis, expression of Angpt2443 caused impaired venous development, suggesting enhanced function as a competitive antagonist for Tie2. In mammary gland tumor models, Angpt2443 differentially affected primary tumor growth and vascularization; these varying effects were associated with Angpt2 protein localization in the endothelium or in the stromal extracellular matrix as well as the frequency of Tie2-positive tumor blood vessels. In the presence of metastatic cells, Angpt2443 promoted destabilization of pulmonary vasculature and lung metastasis. In vitro, ANGPT2443 was susceptible to proteolytical cleavage, resulting in a monomeric ligand (ANGPT2DAP) that inhibited ANGPT1- or ANGPT4-induced TIE2 activation but did not bind to alternative ANGPT2 receptor α5ß1 integrin. Collectively, these data reveal novel roles for the ANGPT2 N-terminal domain in blood vessel remodeling, tumor growth, metastasis, integrin binding, and proteolytic regulation. SIGNIFICANCE: This study identifies the role of the N-terminal oligomerization domain of angiopoietin-2 in vascular remodeling and lung metastasis and provides new insights into mechanisms underlying the versatile functions of angiopoietin-2 in cancer.See related commentary by Kamiyama and Augustin, p. 35.

EphrinB2-EphB4 signalling provides Rho-mediated homeostatic control of lymphatic endothelial cell junction integrity.

Endothelial integrity is vital for homeostasis and adjusted to tissue demands. Although fluid uptake by lymphatic capillaries is a critical attribute of the lymphatic vasculature, the barrier function of collecting lymphatic vessels is also important by ensuring efficient fluid drainage as well as lymph node delivery of antigens and immune cells. Here, we identified the transmembrane ligand EphrinB2 and its receptor EphB4 as critical homeostatic regulators of collecting lymphatic vessel integrity. Conditional gene deletion in mice revealed that EphrinB2/EphB4 signalling is dispensable for blood endothelial barrier function, but required for stabilization of lymphatic endothelial cell (LEC) junctions in different organs of juvenile and adult mice. Studies in primary human LECs further showed that basal EphrinB2/EphB4 signalling controls junctional localisation of the tight junction protein CLDN5 and junction stability via Rac1/Rho-mediated regulation of cytoskeletal contractility. EphrinB2/EphB4 signalling therefore provides a potential therapeutic target to selectively modulate lymphatic vessel permeability and function.

Lymph vessels are thin walled tubes that, similar to blood vessels, carry white blood cells, fluids and waste. Unlike veins and arteries, however, lymph vessels do not carry red blood cells and their main function is to remove excess fluid from tissues. The cells that line vessels in the body are called endothelial cells, and they are tightly linked together by proteins to control what goes into and comes out of the vessels. The chemical, physical and mechanical signals that control the junctions between endothelial cells are often the same in different vessel types, but their effects can vary. The endothelial cells of both blood and lymph vessels have two interacting proteins on their membrane known as EphrinB2 and its receptor, EphB4. When these two proteins interact, the EphB4 receptor becomes activated, which leads to changes in the junctions that link endothelial cells together. Frye et al. examined the role of EphrinB2 and EphB4 in the lymphatic system of mice. When either EphrinB2 or EphB4 are genetically removed in newborn or adult mice, lymph vessels become disrupted, but no significant effect is observed on blood vessels. The reason for the different responses in blood and lymph vessels is unknown. The results further showed that lymphatic endothelial cells need EphB4 and EphrinB2 to be constantly interacting to maintain the integrity of the lymph vessels. Further examination of human endothelial cells grown in the laboratory revealed that this constant signalling controls the internal protein scaffold that determines a cell's shape and integrity. Changes in the internal scaffold affect the organization of the junctions that link neighboring lymphatic endothelial cells together. The loss of signalling between EphrinB2 and EphB4 in lymph vessels reflects the increase in vessel leakage seen in response to bacterial infections and in some genetic conditions such as lymphoedema. Finding ways to control the signalling between these two proteins could help treat these conditions by developing drugs that improve endothelial cell integrity in lymph vessels.

Feasibility of MRI-guided transurethral ultrasound for lesion-targeted ablation of prostate cancer.

Background: MRI-guided transurethral ultrasound ablation (TULSA) has been evaluated for organ-confined prostate cancer (PCa). The purpose of this study was to assess the safety and toxicity, accuracy and short-term evolution of cell-death after lesion-targeted TULSA.Methods: This prospective, registered, Phase-I treat-and-3-week-resect-study enrolled six patients with MRI-visible-biopsy-concordant PCa. Lesions were targeted using TULSA with radical intent, except near neurovascular bundles (NVB). Robot-assisted-laparoscopic-prostatectomy (RALP) was performed at 3 weeks. Post-TULSA assessments included MRI (1 and 3 weeks), adverse events and quality-of-life (QoL) to 3 weeks, followed by RALP and whole-mount-histology. Treatment accuracy and demarcation of thermal injury were assessed using MRI and histology.Results: Six patients (median age = 70 years, prostate volume = 60 ml, PSA = 8.9 ng/ml) with eight biopsy-confirmed MRI-lesions (PIRADS ≥3) were TULSA-treated without complications (median sonication and MRI-times of 17 and 117 min). Foley-catheter removal was uneventful at 2-3 days. Compared to baseline, no differences in QoL were noted at 3 weeks. During follow-up, MRI-derived non-perfused-volume covered ablated targets and increased 36% by 3 weeks, correlating with necrosis-area on histology. Mean histological demarcation between complete necrosis and outer-limit-of-thermal-injury was 1.7 ± 0.4 mm. Coagulation necrosis extended to capsule except near NVB, where 3 mm safety-margins were applied. RALPs were uncomplicated and histopathology showed no viable cancer within the ablated tumor-containing target.Conclusions: Lesion-targeted TULSA demonstrates accurate and safe ablation of PCa. A significant increase of post-TULSA non-perfused-volume was observed during 3 weeks follow-up concordant with necrosis on histology. TULSA achieved coagulation necrosis of all targeted tissues. A limitation of this treat-and-resect-study-design was conservative treatment near NVB in patients scheduled for RALP.

Histopathological evaluation of prostate specimens after thermal ablation may be confounded by the presence of thermally-fixed cells.

Purpose: Prostate cancer can be eradicated with heat exposure. However, high and rapid temperature elevations may cause thermofixation giving the appearance of viable tissue. The purpose was to characterize the immunoprofile and evaluate the viability of prostate regions with suspected thermofixation. Methods and materials: A prospective, ethics-approved and registered study (NCT03350529) enrolled six patients with MRI-visible, biopsy-concordant prostate cancer to undergo lesion-targeted MRI-guided transurethral ultrasound ablation (TULSA) followed by radical prostatectomy at 3 weeks, to evaluate the accuracy and efficacy of TULSA with whole-mount histology as a reference standard. If ambiguity about complete necrosis within the ablated region remained after hematoxylin-eosin staining, viability was assessed by immunohistochemistry. Treatment day MRI-thermometry and 3-week contrast-enhanced MRI post-TULSA were examined to assess ablation success and correlation with histopathology. Results: One patient presented with an apparently viable subregion inside the ablated area, surrounded by necrosis on H&E staining, located where temperature was highest on MRI-thermometry and tissues completely devascularized on MRI. Immunoprofile of the apparently viable tissue revealed changes in staining patterns suggesting thermofixation; the most significant evidence was the negative cytokeratin 8 staining detected with Cam5.2 antibody. A comprehensive literature review supports these observations of thermofixation with similar findings in prostate and other tissues. Conclusion: Thermally-fixed cells can sustain morphology on H&E staining. Misinterpretation of treatment failure may occur, if this phenomenon is not recognized and immunohistochemistry performed. Based on the previous literature and the current study, Cam5.2 staining for cytokeratin 8 appears to be a practical and reliable tool for distinguishing thermally-fixed from viable cells.

IMPROD biparametric MRI in men with a clinical suspicion of prostate cancer (IMPROD Trial): Sensitivity for prostate cancer detection in correlation with whole-mount prostatectomy sections and implications for focal therapy.

BACKGROUND: Prostate MRI is increasingly being used in men with a clinical suspicion of prostate cancer (PCa). However, development and validation of methods for focal therapy planning are still lagging. PURPOSE: To evaluate the diagnostic accuracy on lesion, region-of-interest (ROI), and voxel level of IMPROD biparametric prostate MRI (bpMRI) for PCa detection in men with a clinical suspicion of PCa who subsequently underwent radical prostatectomy. STUDY TYPE: Prospective single-institution clinical trial (NCT01864135). POPULATION: Sixty-four men who underwent radical prostatectomy after IMPROD bpMRI performed in prebiopsy settings. FIELD STRENGTH/SEQUENCE: IMPROD bpMRI consisted of T2 -weighted imaging (T2 w) and three separate diffusion-weighted imaging acquisitions with an average acquisition time of 15 minutes. ASSESSMENT: The diagnostic accuracy of prospectively reported manual cancer delineations and regions increased with 3D dilation were evaluated on the voxel level (volume of 1.17 mm3 , 1 mm3 , 125 mm3 ) as well as the 36 ROI level. Only PCa lesions with a diameter ≥ 5 mm or any Gleason Grade 4 were analyzed. All data and protocols are freely available at: http://petiv.utu.fi/improd STATISTICAL TESTS: Sensitivity, specificity, accuracy. RESULTS: In total, 99 PCa lesions were identified. Forty (40%, 40/99) had a Gleason score (GS) of >3 + 4. Twenty-eight PCa lesions (28%, 28/99) were missed by IMPROD bpMRI, three (7.5%, 3/40) with GS >3 + 4. 3D dilation of manual cancer delineations in all directions by ~10-12 mm (corresponding to the Hausdorff distance) was needed to achieve sensitivity approaching 100% on a voxel level. DATA CONCLUSION: IMPROD bpMRI had a high sensitivity on lesion level for PCa with GS >3 + 4. Increasing 3D lesion delineations by ~10-12 mm (corresponding to the Hausdorff distance) was needed to achieve high sensitivity on the voxel level. Such information may help in planning ablation therapies. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2019;50:1641-1650.

Development of Molecular Therapies for Venous Malformations.

Vascular anomalies are localized defects of morphogenesis that can affect lymphatic and blood vessels. They are generally called birthmarks, typically observed soon after birth and occurring in up to 10% of children. Based on their clinical and histological characteristics, they are classified into vascular tumours and vascular malformations. The most common malformations are venous malformations (VMs) resulting in chronic vascular diseases that can be associated with significant morbidity necessitating often demanding and repeating clinical management. The current treatment is based on surgical resection and sclerotherapy, which can be impossible due to the size or location of lesions or ineffective due to the regrowth of malformed vessels. Therefore, medical therapies for VMs are highly desired. Recent studies have identified genetic defects that result in the constantly active endothelial cell receptor tyrosine kinase TIE2/phosphoinositide 3-kinase PI3K signalling pathway as a frequent cause for VMs. The first treatment to inhibit this pathway with sirolimus indicated that molecular treatment can be effective against VMs. In addition, certain VM 'hotspot' mutations have been previously found in tumours, providing the rationale for the exploration and repurposing of existing and investigational cancer drugs for VMs. Finally, discoveries of molecular and cellular abnormalities that characterize a large proportion of VMs and the generation of pre-clinical VM mouse models provide the necessary basis for the development of the targeted molecular treatment strategies we discuss in this MiniReview.

Therapeutic targeting of the angiopoietin-TIE pathway.

The endothelial angiopoietin (ANG)-TIE growth factor receptor pathway regulates vascular permeability and pathological vascular remodelling during inflammation, tumour angiogenesis and metastasis. Drugs that target the ANG-TIE pathway are in clinical development for oncological and ophthalmological applications. The aim is to complement current vascular endothelial growth factor (VEGF)-based anti-angiogenic therapies in cancer, wet age-related macular degeneration and macular oedema. The unique function of the ANG-TIE pathway in vascular stabilization also renders this pathway an attractive target in sepsis, organ transplantation, atherosclerosis and vascular complications of diabetes. This Review covers key aspects of the function of the ANG-TIE pathway in vascular disease and describes the recent development of novel therapeutics that target this pathway.

VEGFR3 Modulates Vascular Permeability by Controlling VEGF/VEGFR2 Signaling.

RATIONALE: Vascular endothelial growth factor (VEGF) is the main driver of angiogenesis and vascular permeability via VEGF receptor 2 (VEGFR2), whereas lymphangiogenesis signals are transduced by VEGFC/D via VEGFR3. VEGFR3 also regulates sprouting angiogenesis and blood vessel growth, but to what extent VEGFR3 signaling controls blood vessel permeability remains unknown. OBJECTIVE: To investigate the role of VEGFR3 in the regulation of VEGF-induced vascular permeability. METHODS AND RESULTS: Long-term global Vegfr3 gene deletion in adult mice resulted in increased fibrinogen deposition in lungs and kidneys, indicating enhanced vascular leakage at the steady state. Short-term deletion of Vegfr3 in blood vascular endothelial cells increased baseline leakage in various tissues, as well as in tumors, and exacerbated vascular permeability in response to VEGF, administered via intradermal adenoviral delivery or through systemic injection of recombinant protein. VEGFR3 gene silencing upregulated VEGFR2 protein levels and phosphorylation in cultured endothelial cells. Consistent with elevated VEGFR2 activity, vascular endothelial cadherin showed reduced localization at endothelial cell-cell junctions in postnatal retinas after Vegfr3 deletion, or after VEGFR3 silencing in cultured endothelial cells. Furthermore, concurrent deletion of Vegfr2 prevented VEGF-induced excessive vascular leakage in mice lacking Vegfr3. CONCLUSIONS: VEGFR3 limits VEGFR2 expression and VEGF/VEGFR2 pathway activity in quiescent and angiogenic blood vascular endothelial cells, thereby preventing excessive vascular permeability.

Angiopoietin-Tie signalling in the cardiovascular and lymphatic systems.

Endothelial cells that form the inner layer of blood and lymphatic vessels are important regulators of vascular functions and centrally involved in the pathogenesis of vascular diseases. In addition to the vascular endothelial growth factor (VEGF) receptor pathway, the angiopoietin (Ang)-Tie system is a second endothelial cell specific ligand-receptor signalling system necessary for embryonic cardiovascular and lymphatic development. The Ang-Tie system also regulates postnatal angiogenesis, vessel remodelling, vascular permeability and inflammation to maintain vascular homoeostasis in adult physiology. This system is implicated in numerous diseases where the vasculature has an important contribution, such as cancer, sepsis, diabetes, atherosclerosis and ocular diseases. Furthermore, mutations in the TIE2 signalling pathway cause defects in vascular morphogenesis, resulting in venous malformations and primary congenital glaucoma. Here, we review recent advances in the understanding of the Ang-Tie signalling system, including cross-talk with the vascular endothelial protein tyrosine phosphatase (VE-PTP) and the integrin cell adhesion receptors, focusing on the Ang-Tie system in vascular development and pathogenesis of vascular diseases.

Blue Rubber Bleb Nevus (BRBN) Syndrome Is Caused by Somatic TEK (TIE2) Mutations.

Blue rubber bleb nevus syndrome (Bean syndrome) is a rare, severe disorder of unknown cause, characterized by numerous cutaneous and internal venous malformations; gastrointestinal lesions are pathognomonic. We discovered somatic mutations in TEK, the gene encoding TIE2, in 15 of 17 individuals with blue rubber bleb nevus syndrome. Somatic mutations were also identified in five of six individuals with sporadically occurring multifocal venous malformations. In contrast to common unifocal venous malformation, which is most often caused by the somatic L914F TIE2 mutation, multifocal forms are predominantly caused by double (cis) mutations, that is, two somatic mutations on the same allele of the gene. Mutations are identical in all lesions from a given individual. T1105N-T1106P is recurrent in blue rubber bleb nevus, whereas Y897C-R915C is recurrent in sporadically occurring multifocal venous malformation: both cause ligand-independent activation of TIE2, and increase survival, invasion, and colony formation when expressed in human umbilical vein endothelial cells.

VEGFB/VEGFR1-Induced Expansion of Adipose Vasculature Counteracts Obesity and Related Metabolic Complications.

Impaired angiogenesis has been implicated in adipose tissue dysfunction and the development of obesity and associated metabolic disorders. Here, we report the unexpected finding that vascular endothelial growth factor B (VEGFB) gene transduction into mice inhibits obesity-associated inflammation and improves metabolic health without changes in body weight or ectopic lipid deposition. Mechanistically, the binding of VEGFB to VEGF receptor 1 (VEGFR1, also known as Flt1) activated the VEGF/VEGFR2 pathway and increased capillary density, tissue perfusion, and insulin supply, signaling, and function in adipose tissue. Furthermore, endothelial Flt1 gene deletion enhanced the effect of VEGFB, activating the thermogenic program in subcutaneous adipose tissue, which increased the basal metabolic rate, thus preventing diet-induced obesity and related metabolic complications. In obese and insulin-resistant mice, Vegfb gene transfer, together with endothelial Flt1 gene deletion, induced weight loss and mitigated the metabolic complications, demonstrating the therapeutic potential of the VEGFB/VEGFR1 pathway.

A novel human leiomyoma tissue derived matrix for cell culture studies.

BACKGROUND: The composition of the matrix molecules is important in in vitro cell culture experiments of e.g. human cancer invasion and vessel formation. Currently, the mouse Engelbreth-Holm-Swarm (EHS) sarcoma-derived products, such as Matrigel®, are the most commonly used tumor microenvironment (TME) mimicking matrices for experimental studies. However, since Matrigel® is non-human in origin, its molecular composition does not accurately simulate human TME. We have previously described a solid 3D organotypic myoma disc invasion assay, which is derived from human uterus benign leiomyoma tumor. Here, we describe the preparation and analyses of a processed, gelatinous leiomyoma matrix, named Myogel. METHODS: A total protein extract, Myogel, was formulated from myoma. The protein contents of Myogel were characterized and its composition and properties compared with a commercial mouse Matrigel®. Myogel was tested and compared to Matrigel® in human cell adhesion, migration, invasion, colony formation, spheroid culture and vessel formation experiments, as well as in a 3D hanging drop video image analysis. RESULTS: We demonstrated that only 34% of Myogel's molecular content was similar to Matrigel®. All test results showed that Myogel was comparable with Matrigel®, and when mixed with low-melting agarose (Myogel-LMA) it was superior to Matrigel® in in vitro Transwell® invasion and capillary formation assays. CONCLUSIONS: In conclusion, we have developed a novel Myogel TME matrix, which is recommended for in vitro human cell culture experiments since it closely mimics the human tumor microenvironment of solid cancers.

Somatic Activating PIK3CA Mutations Cause Venous Malformation.

Somatic mutations in TEK, the gene encoding endothelial cell tyrosine kinase receptor TIE2, cause more than half of sporadically occurring unifocal venous malformations (VMs). Here, we report that somatic mutations in PIK3CA, the gene encoding the catalytic p110α subunit of PI3K, cause 54% (27 out of 50) of VMs with no detected TEK mutation. The hotspot mutations c.1624G>A, c.1633G>A, and c.3140A>G (p.Glu542Lys, p.Glu545Lys, and p.His1047Arg), frequent in PIK3CA-associated cancers, overgrowth syndromes, and lymphatic malformation (LM), account for >92% of individuals who carry mutations. Like VM-causative mutations in TEK, the PIK3CA mutations cause chronic activation of AKT, dysregulation of certain important angiogenic factors, and abnormal endothelial cell morphology when expressed in human umbilical vein endothelial cells (HUVECs). The p110α-specific inhibitor BYL719 restores all abnormal phenotypes tested, in PIK3CA- as well as TEK-mutant HUVECs, demonstrating that they operate via the same pathogenic pathways. Nevertheless, significant genotype-phenotype correlations in lesion localization and histology are observed between individuals with mutations in PIK3CA versus TEK, pointing to gene-specific effects.

Automated tracking of tumor-stroma morphology in microtissues identifies functional targets within the tumor microenvironment for therapeutic intervention.

Cancer-associated fibroblasts (CAFs) constitute an important part of the tumor microenvironment and promote invasion via paracrine functions and physical impact on the tumor. Although the importance of including CAFs into three-dimensional (3D) cell cultures has been acknowledged, computational support for quantitative live-cell measurements of complex cell cultures has been lacking. Here, we have developed a novel automated pipeline to model tumor-stroma interplay, track motility and quantify morphological changes of 3D co-cultures, in real-time live-cell settings. The platform consists of microtissues from prostate cancer cells, combined with CAFs in extracellular matrix that allows biochemical perturbation. Tracking of fibroblast dynamics revealed that CAFs guided the way for tumor cells to invade and increased the growth and invasiveness of tumor organoids. We utilized the platform to determine the efficacy of inhibitors in prostate cancer and the associated tumor microenvironment as a functional unit. Interestingly, certain inhibitors selectively disrupted tumor-CAF interactions, e.g. focal adhesion kinase (FAK) inhibitors specifically blocked tumor growth and invasion concurrently with fibroblast spreading and motility. This complex phenotype was not detected in other standard in vitro models. These results highlight the advantage of our approach, which recapitulates tumor histology and can significantly improve cancer target validation in vitro.

Rapamycin improves TIE2-mutated venous malformation in murine model and human subjects.

Venous malformations (VMs) are composed of ectatic veins with scarce smooth muscle cell coverage. Activating mutations in the endothelial cell tyrosine kinase receptor TIE2 are a common cause of these lesions. VMs cause deformity, pain, and local intravascular coagulopathy, and they expand with time. Targeted pharmacological therapies are not available for this condition. Here, we generated a model of VMs by injecting HUVECs expressing the most frequent VM-causing TIE2 mutation, TIE2-L914F, into immune-deficient mice. TIE2-L914F-expressing HUVECs formed VMs with ectatic blood-filled channels that enlarged over time. We tested both rapamycin and a TIE2 tyrosine kinase inhibitor (TIE2-TKI) for their effects on murine VM expansion and for their ability to inhibit mutant TIE2 signaling. Rapamycin prevented VM growth, while TIE2-TKI had no effect. In cultured TIE2-L914F-expressing HUVECs, rapamycin effectively reduced mutant TIE2-induced AKT signaling and, though TIE2-TKI did target the WT receptor, it only weakly suppressed mutant-induced AKT signaling. In a prospective clinical pilot study, we analyzed the effects of rapamycin in 6 patients with difficult-to-treat venous anomalies. Rapamycin reduced pain, bleeding, lesion size, functional and esthetic impairment, and intravascular coagulopathy. This study provides a VM model that allows evaluation of potential therapeutic strategies and demonstrates that rapamycin provides clinical improvement in patients with venous malformation.

Endothelial destabilization by angiopoietin-2 via integrin ß1 activation.

Angiopoietins regulate vascular homeostasis via the endothelial Tie receptor tyrosine kinases. Angiopoietin-1 (Ang1) supports endothelial stabilization via Tie2 activation. Angiopoietin-2 (Ang2) functions as a context-dependent Tie2 agonist/antagonist promoting pathological angiogenesis, vascular permeability and inflammation. Elucidating Ang2-dependent mechanisms of vascular destablization is critical for rational design of angiopoietin antagonists that have demonstrated therapeutic efficacy in cancer trials. Here, we report that Ang2, but not Ang1, activates ß1-integrin, leading to endothelial destablization. Autocrine Ang2 signalling upon Tie2 silencing, or in Ang2 transgenic mice, promotes ß1-integrin-positive elongated matrix adhesions and actin stress fibres, regulating vascular endothelial-cadherin-containing cell-cell junctions. The Tie2-silenced monolayer integrity is rescued by ß1-integrin, phosphoinositide-3 kinase or Rho kinase inhibition, and by re-expression of a membrane-bound Tie2 ectodomain. Furthermore, Tie2 silencing increases, whereas Ang2 blocking inhibits transendothelial tumour cell migration in vitro. These results establish Ang2-mediated ß1-integrin activation as a promoter of endothelial destablization, explaining the controversial vascular functions of Ang1 and Ang2.