Combining laser microdissection and microRNA expression profiling to unmask microRNA signatures in complex tissues

Neglecting tissue heterogeneity during the analysis of microRNA (miRNA) levels results in average signals from an unknown mixture of different cell types that are difficult to interpret. Here we demonstrate the technical requirements needed to obtain high-quality, quantitative miRNA expression infor- mation from tumor tissue compartments obtained by laser microdissection (LMD). Furthermore, we show the significance of disentangling tumor tissue heterogeneity by applying the newly developed protocols for combining LMD of tumor tissue compartments with RT-qPCR analysis to reveal compartment- specific miRNA expression signatures. An important advantage of this strategy is that the miRNA signature can be directly linked to histopatho logy. In summary, combining LMD and RT-qPCR is a powerful approach for spatial miRNA expression analysis in complex tissues, enabling discovery of disease mechanisms, biomarkers and drug candidates

Pattern of tamoxifen-induced Tie2 deletion in endothelial cells in mature blood vessels using endo SCL-Cre-ERT transgenic mice

Tyrosine-protein kinase receptor Tie2, also known as Tunica interna Endothelial cell Kinase or TEK plays a prominent role in endothelial responses to angiogenic and inflammatory stimuli. Here we generated a novel inducible Tie2 knockout mouse model, which targets mature (micro)vascular endothelium, enabling the study of the organ-specific contribution of Tie2 to these responses. Mice with floxed Tie2 exon 9 alleles (Tie2floxed/floxed) were crossed with end-SCL-Cre-ERT transgenic mice, generating offspring in which Tie2 exon 9 is deleted in the endothelial compartment upon tamoxifen-induced activation of Cre-recombinase (Tie2ΔE9). Successful deletion of Tie2 exon 9 in kidney, lung, heart, aorta, and liver, was accompanied by a heterogeneous, organ-dependent reduction in Tie2 mRNA and protein expression. Microvascular compartment-specific reduction in Tie2 mRNA and protein occurred in arterioles of all studied organs, in renal glomeruli, and in lung capillaries. In kidney, lung, and heart, reduced Tie2 expression was accompanied by a reduction in Tie1 mRNA expression. The heterogeneous, organ- and microvascular compartment-dependent knockout pattern of Tie2 in the Tie2floxed/floxed;end-SCL-Cre-ERT mouse model suggests that future studies using similar knockout strategies should include a meticulous analysis of the knockout extent of the gene of interest, prior to studying its role in pathological conditions, so that proper conclusions can be drawn

Pharmacological inhibition of protein tyrosine kinases axl and fyn reduces TNF-α-induced endothelial inflammatory activation in vitro

Major surgery induces systemic inflammation leading to pro-inflammatory activation of endothelial cells. Endothelial inflammation is one of the drivers of postoperative organ damage, including acute kidney injury Tumour Necrosis Factor alpha (TNF-α) is an important component of surgery-induced pro-inflammatory activation of endothelial cells. Kinases, the backbone of signalling cascades, can be targeted by pharmacological inhibition. This is a promising treatment option to interfere with excessive endothelial inflammation. In this study, we identified activated kinases as potential therapeutic targets. These targets were pharmacologically inhibited to reduce TNF-α-induced pro-inflammatory signalling in endothelial cells. Kinome profiling using PamChip arrays identified 64 protein tyrosine kinases and 88 serine-threonine kinases, the activity of which was determined at various timepoints (5–240 min) following stimulation with 10 ng/ml TNF-α in Human umbilical vein endothelial cells in vitro. The PTKs Axl and Fyn were selected based on high kinase activity profiles. Co-localisation experiments with the endothelial-specific protein CD31 showed Axl expression in endothelial cells of glomeruli and Fyn in arterioles and glomeruli of both control and TNF-α-exposed mice. Pharmacological inhibition with Axl inhibitor BMS-777607 and Fyn inhibitor PP2 significantly reduced TNF-α-induced pro-inflammatory activation of E-selectin, VCAM-1, ICAM-1, IL-6 and IL-8 at mRNA and VCAM-1, ICAM-1, and IL-6 at protein level in HUVEC in vitro. Upon pharmacological inhibition with each inhibitor, leukocyte adhesion to HUVEC was also significantly reduced, however to a minor extent. In conclusion, pre-treatment of endothelial cells with kinase inhibitors BMS-777607 and PP2 reduces TNF-α-induced endothelial inflammation in vitro

H3K27Me3 abundance increases fibrogenesis during endothelial-to-mesenchymal transition via the silencing of microRNA-29c

OBJECTIVE: Endothelial-to-mesenchymal transition (EndMT) is a transdifferentiation process in which endothelial cells (ECs) adopt a mesenchymal-like phenotype. Over the past few years, it became clear that EndMT can contribute to several cardiovascular pathologies. However, the molecular pathways underlying the development of EndMT remain incompletely understood. Since the epigenetic enzyme Enhancer of Zeste Homolog 2 (EZH2) and its concomitant mark H3K27Me3 have been shown to be elevated in many cardiovascular diseases that associate with EndMT, we hypothesized that H3K27Me3 is a determinant for the susceptibility of EndMT.METHODS: To study the association between H3K27Me3 and EndMT, a knockdown model of EZH2 in human endothelial cells (HUVEC) was utilized to reduce H3K27Me3 abundance, followed by induction of EndMT using TGFβ1. The expression of molecular markers of EndMT and fibrogenesis were analysed.RESULTS: In cultured HUVECs, a reduction of H3K27Me3 abundance facilitates EndMT but mitigates fibrogenesis as shown by a decreased expression of collagen I and III. In HUVEC, H3K27Me3 abundance directly affects the expression of miR29c, a collagen-targeting miRNA. Additionally, knockdown of miR-29c in HUVEC with low H3K27Me3 abundance partly restored the expression of collagen I and III. Expectedly, in rats with perivascular fibrosis an increased abundance of H3K27Me3 associated with a decreased expression of miR-29c.CONCLUSION: our data shows that endothelial fibrogenesis underlies an epigenetic regulatory pathway and we demonstrate that a decreased abundance of H3K27Me3 in ECs blunts fibrogenesis in part in a miR-29c dependent manner. Therefore, a reduction of H3K27Me3 could serve as a novel therapeutical strategy to mitigate fibrogenesis and may prove to be beneficial in fibrogenic diseases including atherosclerosis, cardiac fibrosis, and PAH.</p

Pharmacological inhibition of focal adhesion kinase 1 (FAK1) and anaplastic lymphoma kinase (ALK) identified via kinome profile analysis attenuates lipopolysaccharide-induced endothelial inflammatory activation

Sepsis is a life-threatening condition often leading to multiple organ failure for which currently no pharmacological treatment is available. Endothelial cells (EC) are among the first cells to respond to pathogens and inflammatory mediators in sepsis and might be a sentinel target to prevent the occurrence of multiple organ failure. Lipopolysaccharide (LPS) is a Gram-negative bacterial component that induces endothelial expression of inflammatory adhesion molecules, cytokines, and chemokines. This expression is regulated by a network of kinases, the result of which in vivo enables leukocytes to transmigrate from the blood into the underlying tissue, causing organ damage. We hypothesised that besides the known kinase pathways, other kinases are involved in the regulation of EC in response to LPS, and that these can be pharmacologically targeted to inhibit cell activation. Using kinome profiling, we identified 58 tyrosine kinases (TKs) that were active in human umbilical vein endothelial cells (HUVEC) at various timepoints after stimulation with LPS. These included AXL tyrosine kinase (Axl), focal adhesion kinase 1 (FAK1), and anaplastic lymphoma kinase (ALK). Using siRNA-based gene knock down, we confirmed that these three TKs mediate LPS-induced endothelial inflammatory activation. Pharmacological inhibition with FAK1 inhibitor FAK14 attenuated LPS-induced endothelial inflammatory activation and leukocyte adhesion partly via blockade of NF-κB activity. Administration of FAK14 after EC exposure to LPS also resulted in inhibition of inflammatory molecule expression. In contrast, inhibition of ALK with FDA-approved inhibitor Ceritinib attenuated LPS-induced endothelial inflammatory activation via a pathway that was independent of NF-κB signalling while it did not affect leukocyte adhesion. Furthermore, Ceritinib administration after start of EC exposure to LPS did not inhibit inflammatory activation. Combined FAK1 and ALK inhibition attenuated LPS-induced endothelial activation in an additive manner, without affecting leukocyte adhesion. Summarising, our findings suggest the involvement of FAK1 and ALK in mediating LPS-induced inflammatory activation of EC. Since pharmacological inhibition of FAK1 attenuated endothelial inflammatory activation after the cells were exposed to LPS, FAK1 represents a promising target for follow up studies

Pharmacological inhibition of protein tyrosine kinases axl and fyn reduces TNF-α-induced endothelial inflammatory activation in vitro

Major surgery induces systemic inflammation leading to pro-inflammatory activation of endothelial cells. Endothelial inflammation is one of the drivers of postoperative organ damage, including acute kidney injury Tumour Necrosis Factor alpha (TNF-α) is an important component of surgery-induced pro-inflammatory activation of endothelial cells. Kinases, the backbone of signalling cascades, can be targeted by pharmacological inhibition. This is a promising treatment option to interfere with excessive endothelial inflammation. In this study, we identified activated kinases as potential therapeutic targets. These targets were pharmacologically inhibited to reduce TNF-α-induced pro-inflammatory signalling in endothelial cells. Kinome profiling using PamChip arrays identified 64 protein tyrosine kinases and 88 serine-threonine kinases, the activity of which was determined at various timepoints (5–240 min) following stimulation with 10 ng/ml TNF-α in Human umbilical vein endothelial cells in vitro. The PTKs Axl and Fyn were selected based on high kinase activity profiles. Co-localisation experiments with the endothelial-specific protein CD31 showed Axl expression in endothelial cells of glomeruli and Fyn in arterioles and glomeruli of both control and TNF-α-exposed mice. Pharmacological inhibition with Axl inhibitor BMS-777607 and Fyn inhibitor PP2 significantly reduced TNF-α-induced pro-inflammatory activation of E-selectin, VCAM-1, ICAM-1, IL-6 and IL-8 at mRNA and VCAM-1, ICAM-1, and IL-6 at protein level in HUVEC in vitro. Upon pharmacological inhibition with each inhibitor, leukocyte adhesion to HUVEC was also significantly reduced, however to a minor extent. In conclusion, pre-treatment of endothelial cells with kinase inhibitors BMS-777607 and PP2 reduces TNF-α-induced endothelial inflammation in vitro

Pharmacological inhibition of protein tyrosine kinases axl and fyn reduces TNF-α-induced endothelial inflammatory activation in vitro

Major surgery induces systemic inflammation leading to pro-inflammatory activation of endothelial cells. Endothelial inflammation is one of the drivers of postoperative organ damage, including acute kidney injury Tumour Necrosis Factor alpha (TNF-α) is an important component of surgery-induced pro-inflammatory activation of endothelial cells. Kinases, the backbone of signalling cascades, can be targeted by pharmacological inhibition. This is a promising treatment option to interfere with excessive endothelial inflammation. In this study, we identified activated kinases as potential therapeutic targets. These targets were pharmacologically inhibited to reduce TNF-α-induced pro-inflammatory signalling in endothelial cells. Kinome profiling using PamChip arrays identified 64 protein tyrosine kinases and 88 serine-threonine kinases, the activity of which was determined at various timepoints (5–240 min) following stimulation with 10 ng/ml TNF-α in Human umbilical vein endothelial cells in vitro. The PTKs Axl and Fyn were selected based on high kinase activity profiles. Co-localisation experiments with the endothelial-specific protein CD31 showed Axl expression in endothelial cells of glomeruli and Fyn in arterioles and glomeruli of both control and TNF-α-exposed mice. Pharmacological inhibition with Axl inhibitor BMS-777607 and Fyn inhibitor PP2 significantly reduced TNF-α-induced pro-inflammatory activation of E-selectin, VCAM-1, ICAM-1, IL-6 and IL-8 at mRNA and VCAM-1, ICAM-1, and IL-6 at protein level in HUVEC in vitro. Upon pharmacological inhibition with each inhibitor, leukocyte adhesion to HUVEC was also significantly reduced, however to a minor extent. In conclusion, pre-treatment of endothelial cells with kinase inhibitors BMS-777607 and PP2 reduces TNF-α-induced endothelial inflammation in vitro

Raw data - Unique miRNome and Transcriptome Profiles Underlie Microvascular Heterogeneity in Mouse Kidney

Raw data of the manuscript "Unique miRNome and Transcriptome Profiles Underlie Microvascular Heterogeneity in Mouse Kidney" is included. Types of included data are - NDPI files showing immunohistochemistry or in situ hybridisation in mouse kidney - Differential expression analysis data from both small RNA sequencing and RNA sequencing - Raw CT values of all included RT-qPCRs</p