Off-targets of BRAF inhibitors disrupt endothelial signaling and vascular barrier function.

Targeted therapies against mutant BRAF are effectively used in combination with MEK inhibitors (MEKi) to treat advanced melanoma. However, treatment success is affected by resistance and adverse events (AEs). Approved BRAF inhibitors (BRAFi) show high levels of target promiscuity, which can contribute to these effects. The blood vessel lining is in direct contact with high plasma concentrations of BRAFi, but effects of the inhibitors in this cell type are unknown. Hence, we aimed to characterize responses to approved BRAFi for melanoma in the vascular endothelium. We showed that clinically approved BRAFi induced a paradoxical activation of endothelial MAPK signaling. Moreover, phosphoproteomics revealed distinct sets of off-targets per inhibitor. Endothelial barrier function and junction integrity were impaired upon treatment with vemurafenib and the next-generation dimerization inhibitor PLX8394, but not with dabrafenib or encorafenib. Together, these findings provide insights into the surprisingly distinct side effects of BRAFi on endothelial signaling and functionality. Better understanding of off-target effects could help to identify molecular mechanisms behind AEs and guide the continued development of therapies for BRAF-mutant melanoma.

Paracrine Factors of Stressed Peripheral Blood Mononuclear Cells Activate Proangiogenic and Anti-Proteolytic Processes in Whole Blood Cells and Protect the Endothelial Barrier.

Tissue-regenerative properties have been attributed to secreted paracrine factors derived from stem cells and other cell types. In particular, the secretome of γ-irradiated peripheral blood mononuclear cells (PBMCsec) has been shown to possess high tissue-regenerative and proangiogenic capacities in a variety of preclinical studies. In light of future therapeutic intravenous applications of PBMCsec, we investigated the possible effects of PBMCsec on white blood cells and endothelial cells lining the vasculature. To identify changes in the transcriptional profile, whole blood was drawn from healthy individuals and stimulated with PBMCsec for 8 h ex vivo before further processing for single-cell RNA sequencing. PBMCsec significantly altered the gene signature of granulocytes (17 genes), T-cells (45 genes), B-cells (72 genes), and, most prominently, monocytes (322 genes). We detected a strong upregulation of several tissue-regenerative and proangiogenic cyto- and chemokines in monocytes, including VEGFA, CXCL1, and CXCL5. Intriguingly, inhibitors of endopeptidase activity, such as SERPINB2, were also strongly induced. Measurement of the trans-endothelial electrical resistance of primary human microvascular endothelial cells revealed a strong barrier-protective effect of PBMCsec after barrier disruption. Together, we show that PBMCsec induces angiogenic and proteolytic processes in the blood and is able to attenuate endothelial barrier damage. These regenerative properties suggest that systemic application of PBMCsec might be a promising novel strategy to restore damaged organs.

More than Just a Monolayer: the Multifaceted Role of Endothelial Cells in the Pathophysiology of Atherosclerosis.

PURPOSE OF THE REVIEW: In this review, we summarize current insights into the versatile roles of endothelial cells in atherogenesis. RECENT FINDINGS: The vascular endothelium represents the first barrier that prevents the entry of lipoproteins and leukocytes into the vessel wall, thereby controlling two key events in the pathogenesis of atherosclerosis. Disturbance of endothelial homeostasis increases vascular permeability, inflammation, and cellular trans-differentiation, which not only promotes the build-up of atherosclerotic plaques but is also involved in life-threatening thromboembolic complications such as plaque rupture and erosion. In this review, we focus on recent findings on endothelial lipoprotein transport, inflammation, cellular transitions, and barrier function. By using cutting-edge technologies such as single-cell sequencing, epigenetics, and cell fate mapping, novel regulatory mechanisms and endothelial cell phenotypes have been discovered, which have not only challenged established concepts of endothelial activation, but have also led to a different view of the disease.

EVI1 Promotes the Proliferation and Invasive Properties of Human Head and Neck Squamous Cell Carcinoma Cells.

Head and neck squamous cell carcinoma (HNSCC) is a frequent malignancy with a poor prognosis. So far, the EGFR inhibitor cetuximab is the only approved targeted therapy. A deeper understanding of the molecular and genetic basis of HNSCC is needed to identify additional targets for rationally designed, personalized therapeutics. The transcription factor EVI1, the major product of the MECOM locus, is an oncoprotein with roles in both hematological and solid tumors. In HNSCC, high EVI1 expression was associated with an increased propensity to form lymph node metastases, but its effects in this tumor entity have not yet been determined experimentally. We therefore overexpressed or knocked down EVI1 in several HNSCC cell lines and determined the impact of these manipulations on parameters relevant to tumor growth and invasiveness, and on gene expression patterns. Our results revealed that EVI1 promoted the proliferation and migration of HNSCC cells. Furthermore, it augmented tumor spheroid formation and the ability of tumor spheroids to displace an endothelial cell layer. Finally, EVI1 altered the expression of numerous genes in HNSCC cells, which were enriched for Gene Ontology terms related to its cellular functions. In summary, EVI1 represents a novel oncogene in HNSCC that contributes to cellular proliferation and invasiveness.

Phosphorylated cingulin localises GEF-H1 at tight junctions to protect vascular barriers in blood endothelial cells.

Dysfunction of vascular barriers is a critical step in inflammatory diseases. Endothelial tight junctions (TJs) control barrier function, and the cytoplasmic adaptor protein cingulin connects TJs to signalling pathways. However, local events at TJs during inflammation are largely unknown. In this study, we investigate the local response of TJ adaptor protein cingulin and its interaction with Rho guanine nucleotide exchange factor H1 (GEF-H1, also known as ARHGEF2) upon vascular barrier disruption to find a new approach to counteract vascular leak. Based on transendothelial-electrical-resistance (TEER) measurements, cingulin strengthened barrier integrity upon stimulation with histamine, thrombin and VEGF. Cingulin also attenuated myosin light chain 2 (MLC2; also known as MYL2) phosphorylation by localising GEF-H1 to cell junctions. By using cingulin phosphomutants, we verified that the phosphorylation of the cingulin head domain is required for its protective effect. Increased colocalisation of GEF-H1 and cingulin was observed in the vessels of vasculitis patients compared to those in healthy skin. Our findings demonstrate that cingulin can counteract vascular leak at TJs, suggesting the existence of a novel mechanism in blood endothelial cells that protects barrier function during disease.

Synthetic Fibrin-Derived Bß15-42 Peptide Delays Thrombus Resolution in a Mouse Model.

[Figure: see text].

The tight junction protein cingulin regulates the vascular response to burn injury in a mouse model.

Edema formation due to the collapse of physiological barriers and the associated delayed healing process is still a central problem in the treatment of burn injuries. In healthy individuals, tight junctions form a barrier to fluid and small molecules. Cingulin is a cytoplasmic component of tight junctions and is involved in the regulation of the paracellular barrier. Endothelial specific cingulin knock-out mice provide new insight into the influence of tight junction proteins on edema formation and angiogenesis during wound healing. Knock-out mice lacking the head domain of cingulin in endothelial cells (CgnΔEC) were created by breeding Cgnfl/fl mice with Tie1-cre mice. Using a no-touch hot air jet a burn trauma was induced on the ear of the mouse. Over a period of 12 days microcirculatory parameters such as edema formation, angiogenesis and leukocyte-endothelial interactions were visualized using intravital fluorescence microscopy. At baseline, CgnΔEC mice surprisingly showed significantly less tracer extravasation compared to Cgnfl/fl littermates, whereas, after burn injury, edema was consistently higher in CgnΔEC mice. Non-perfused area after wounding was increased, but there was no difference in vessel diameters, contraction or dilation of arteries in CgnΔEC mice. Moreover, cingulin knock-out did not cause a difference in leukocyte adhesion after burn injury. In summary, cingulin limits non-perfused area after burn injury and maintains the paracellular barrier of blood vessels. Since edema formation with serious systemic effects is a central problem of burn wounds, understanding the importance of tight junction proteins might help to find new treatment strategies for burn wounds.

Inhibition of p38/MK2 Signaling Prevents Vascular Invasion of Melanoma.

Melanoma cells can switch between distinct gene expression profiles, resulting in proliferative or invasive phenotypes. Signaling pathways involved in this switch were analyzed by gene expression profiling of a cohort of 22 patient-derived melanoma cell lines. CDH1 negativity was identified as a surrogate marker for the invasive phenotype. CDH1 expression could be turned on and off by modulating activity of p38 or its downstream target MK2, suggesting that this pathway controls melanoma progression. Mechanistically, MK2 inhibition prevented melanoma-induced vascular barrier disruption, reduced the expression of PODXL and DEL-1, and prevented vascular dissemination in vivo. PODXL and DEL-1 expression in patients with melanoma were associated with poor survival and thus can be used as prognostic markers. Downstream targets of MK2 may thus serve as candidate therapeutics.

Differences in biocompatibility of microneedles from cyclic olefin polymers with human endothelial and epithelial skin cells.

Microneedles are promising devices for transdermal delivery and diagnostic applications, due to their minimally invasive and painless nature of application. However, so far, applications are limited to small scale research projects. Material selection and production for larger projects remain a challenge. In vitro testing using human cell culture could bridge the gap between cost effective screening of suitable materials and concerns for safety and ethics. In this study, materials were tested for effects on viability and morphology of human endothelial cells and keratinocytes. In addition, materials were assessed for their potential to influence cellular differentiation and barrier formation. Elution-based testing of inflammatory markers revealed no negative effects in all applied tests, whereas the assessment of differentiation markers on cells in direct contact with the material showed differences and allowed the selection of candidate materials for future medical device applications. This study illustrates that elution-based biocompatibility testing can paint an incomplete picture. Advanced staining techniques and cell types specific for the application of the medical device improve material selection to reduce and replace animal testing at an early stage in the development process. © 2018 The Authors. journal Of Biomedical Materials Research Part A Published By Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 505-512, 2019.

CXCL5 Facilitates Melanoma Cell-Neutrophil Interaction and Lymph Node Metastasis.

Chemokines influence tumor metastasis by targeting tumor, stromal, and hematopoietic cells. Characterizing the chemokine mRNA expression profile of human primary melanoma samples, we found CXCL5 significantly up-regulated in stage T4 primary melanomas when compared to thin melanomas (T1 stage). To characterize the role of CXCL5 in melanoma progression, we established a metastasizing murine xenograft model using CXCL5-overexpressing human melanoma cells. CXCL5 had no effect on melanoma proliferation in vitro and on primary tumor growth in vivo, but CXCL5-overexpressing tumors recruited high amounts of neutrophils and exhibited significantly increased lymphangiogenesis in our severe combined immune-deficient mouse model. Recruited neutrophils were found in close proximity to or within lymphatic vessels, often in direct contact with melanoma cells. Clinically, CXCL5-overexpressing melanomas had significantly increased lymph node metastases. We were able to translate these findings to human patient samples and found a positive correlation between CXCL5 expression, numbers of neutrophils in stage T4 primary melanoma, and the occurrence of subsequent locoregional metastasis.

Evidence That Cingulin Regulates Endothelial Barrier Function In Vitro and In Vivo.

OBJECTIVE: Cingulin is a cytoplasmic component of tight junctions. Although modulation of cingulin levels in cultured epithelial model systems has no significant effect on barrier function, evidence from cingulin knockout mice suggests that cingulin may be involved in the regulation of the behavior of epithelial or endothelial cells. Here, we investigate the role of cingulin in the barrier function of endothelial cells. APPROACH AND RESULTS: We show that cingulin is expressed in human endothelial cells of the skin, brain, and lung in vivo and in vitro. Endothelial cingulin colocalizes and coimmunoprecipitates with the tight junction proteins zonula occludens-1 and guanine nucleotide exchange factor-H1. Cingulin overexpression in human umbilical vein endothelial cell induces tight junction formation, increases transendothelial electric resistance, and strengthens barrier function for low and high molecular weight tracers. In contrast, cultured endothelial cells lacking cingulin are more permeable for low molecular weight tracers. In cingulin knockout mice, neurons of the area postrema and Purkinje cells show an increased uptake of small molecular weight tracers indicating decreased barrier function at these sites. CONCLUSIONS: We demonstrate that cingulin participates in the modulation of endothelial barrier function both in human cultured cells in vitro and in mouse brains in vivo. Understanding the role of cingulin in maintaining tight barriers in endothelia may allow developing new strategies for the treatment of vascular leak syndromes.

A Peptide to Reduce Pulmonary Edema in a Rat Model of Lung Transplantation.

BACKGROUND: Despite significant advances in organ preservation, surgical techniques and perioperative care, primary graft dysfunction is a serious medical problem in transplantation medicine in general and a specific problem in patients undergoing lung transplantation. As a result, patients develop lung edema, causing reduced tissue oxygenation capacity, reduced lung compliance and increased requirements for mechanical ventilatory support. Yet, there is no effective strategy available to protect the grafted organ from stress reactions induced by ischemia/reperfusion and by the surgical procedure itself. METHODS: We assessed the effect of a cingulin-derived peptide, XIB13 or a random peptide in an established rat model of allogeneic lung transplantation. Donor lungs and recipients received therapeutic peptide at the time of transplantation and outcome was analyzed 100min and 28 days post grafting. RESULTS: XIB13 improved blood oxygenation and reduced vascular leak 100min post grafting. Even after 28 days, lung edema was significantly reduced by XIB13 and lungs had reduced fibrotic or necrotic zones. Moreover, the induction of an allogeneic T cell response was delayed indicating a reduced antigen exchange between the donor and the host. CONCLUSIONS: In summary, we provide a new tool to strengthen endothelial barrier function thereby improving outcomes in lung transplantation.

Peptide XIB13 reduces capillary leak in a rodent burn model.

OBJECTIVE: Edema due to capillary leak is a generalized and life threatening event in sepsis and major burns for which there is no causal treatment. Local burn wounds are an ideal model to investigate the impact of a new therapeutic agent on edema formation. We aimed to identify peptide sequences of cingulin that can attenuate stress-induced endothelial cytoskeleton disarrangement in vitro and which reduce burn-induced edema in vivo. METHODS: Cingulin-derived peptides were screened in high content cell culture assays monitoring actin displacement and endothelial cell/cell contacts. The ears of male hairless mice (n=44) were inflicted with full thickness burns using a hot air jet. Mice with and without burn injuries were treated with Xib13 or solvent by continuous intraperitoneal application for 3 days. Edema, microcirculation, leukocyte-endothelial interactions and angiogenesis - measured as non-perfused area - were investigated over a 12-day period using intravital fluorescence microscopy. RESULTS: Xib13 reduced endothelial stress formation and stabilized endothelial tight junctions in cell-cultures. In the burn model, Xib13 improved angiogenesis compared to controls (non-perfused area on day 12: 5.7±1.5% vs. 12.0±2.1%; p<0.05). Edema was significantly reduced at all observation points in Xib13-treated animals as compared to controls (day 12: 67.6±2.6% vs. 83.2±6.4%). CONCLUSION: Xib13 improved angiogenesis, reduced edema formation and showed no side effects on other physiological parameters. Since edema formation is a serious parameter for burn conversion and is associated with survival it could provide a new treatment option for patients with burn injuries.

High thrombin concentrations in fibrin sealants induce apoptosis in human keratinocytes.

Over the last century many studies have been performed to assess the impact of fibrin sealant (FS) components on cells. Because of the noncovalent bonding of thrombin to fibrin during fibrin clot formation, we wanted to further evaluate the impact of fibrin bound thrombin on cell viability. Initially, we quantified the activity of thrombin in three different, commercially available FS. This information was used to prepare fibrin clots covering a range of thrombin concentrations from 4 to 820 IU mL(-1), but which were identical with respect to all other constituents. Although these fibrin clots did not differ in their three-dimensional structure, clots prepared with highly concentrated thrombin (820 IU mL(-1)) failed to support adhesion and spreading of primary human keratinocytes (NHEK). The number of attached cells was also significantly reduced on high thrombin activity clots. We hypothesized that these observations are not only the consequence of decreased proliferation but of apoptotic mechanisms, since the expression of cleaved caspase 3 and 7 was strongly enhanced on fibrin clots with high thrombin activity. This was accompanied by an induction of expression of Trail-R2 which is a receptor known to mediate apoptosis signals. Blocking of thrombin activity by hirudin led to an improvement of cell morphology and to an increase in number of attached cells. In addition, the induction of caspase 3 and 7 was also reduced. Thus, here we report for the first time that fibrin bound thrombin does not only decrease proliferation (as already published by others), it also does induce NHEK apoptosis when present at high concentrations.