7 research outputs found
Inactivation of Cerebral Cavernous Malformation Genes Results in Accumulation of von Willebrand Factor and Redistribution of Weibel-Palade Bodies in Endothelial Cells
Cerebral cavernous malformations are slow-flow thrombi-containing vessels induced by two-step inactivation of the CCM1, CCM2 or CCM3 gene within endothelial cells. They predispose to intracerebral bleedings and focal neurological deficits. Our understanding of the cellular and molecular mechanisms that trigger endothelial dysfunction in cavernous malformations is still incomplete. To model both, hereditary and sporadic CCM disease, blood outgrowth endothelial cells (BOECs) with a heterozygous CCM1 germline mutation and immortalized wild-type human umbilical vein endothelial cells were subjected to CRISPR/Cas9-mediated CCM1 gene disruption. CCM1−/− BOECs demonstrated alterations in cell morphology, actin cytoskeleton dynamics, tube formation, and expression of the transcription factors KLF2 and KLF4. Furthermore, high VWF immunoreactivity was observed in CCM1−/− BOECs, in immortalized umbilical vein endothelial cells upon CRISPR/Cas9-induced inactivation of either CCM1, CCM2 or CCM3 as well as in CCM tissue samples of familial cases. Observer-independent high-content imaging revealed a striking reduction of perinuclear Weibel-Palade bodies in unstimulated CCM1−/− BOECs which was observed in CCM1+/− BOECs only after stimulation with PMA or histamine. Our results demonstrate that CRISPR/Cas9 genome editing is a powerful tool to model different aspects of CCM disease in vitro and that CCM1 inactivation induces high-level expression of VWF and redistribution of Weibel-Palade bodies within endothelial cells
Cerebral arteriovenous malformations : molecular biology and enhancement of radiosurgical treatment
Object
Rupture of intracranial arteriovenous malformations is a leading cause of stroke in children and young adults. Treatment options include surgery and highly focused radiation (stereotactic radiosurgery). For large and deep seated lesions, the risks of surgery may be prohibitively high, while radiosurgery has a disappointingly low efficacy and long latency. Radiosurgery carries the most promise for significant advances, however the process by which radiosurgery achieves obliteration is incompletely understood. Inflammation and thrombosis are likely to be important in the radiation response and may be amenable to pharmacological manipulation to improve radiosurgical efficacy.
Materials and methods
Immunohistochemistry and electron microscopy were used to study normal cerebral vessels, cavernous malformations and AVMs, some of which had previously been irradiated. An attempt was made to culture AVM endothelial cells to study the immediate response of AVM endothelium to radiosurgery. The effects of radiosurgery in a rat model of AVM were studied using immunohistochemistry and the results used to determine the choice of a pharmacological strategy to enhance the thrombotic effects of radiosurgery.
Results
Vascular malformations have a different endothelial inflammatory phenotype than normal cerebral vessels. Radiosurgery may cause long term changes in inflammatory molecule expression and leads to endothelial loss with exposure of pro-thrombotic molecules. Ultrastructural effects of irradiation include widespread cell loss, smooth muscle cell (SMC) proliferation and thrombosis. Endothelial culture from AVMs proved difficult due to SMC predominance in initial cultures.
Radiosurgery upregulated several endothelial inflammatory molecules in the animal model and may induce pro-thrombotic cell membrane alterations. The administration of lipopolysaccharide and soluble tissue factor to rats following radiosurgery led to selective thrombosis of irradiated vessels.
Conclusions
Inflammation and thrombosis are important in the radiosurgical response of AVMs. Lumen obliteration appears to be mediated by proliferation of cells within the vessel wall and thrombosis.
Upregulation of inflammatory molecules and perhaps disruption of the normal phospholipid asymmetry of the endothelial and SMC membranes are some of the earliest responses to radiosurgery. The alterations induced by radiation may be harnessed to selectively initiate thrombus formation. Stimulation of thrombosis may improve the efficacy of radiosurgery, increasing treatable lesion size and reducing latency
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Neuroinflammation Plays a Critical Role in Cerebral Cavernous Malformation Disease
BackgroundCerebral cavernous malformations (CCMs) are neurovascular lesions caused by loss of function mutations in 1 of 3 genes, including KRIT1 (CCM1), CCM2, and PDCD10 (CCM3). CCMs affect ≈1 out of 200 children and adults, and no pharmacologic therapy is available. CCM lesion count, size, and aggressiveness vary widely among patients of similar ages with the same mutation or even within members of the same family. However, what determines the transition from quiescent lesions into mature and active (aggressive) CCM lesions is unknown.MethodsWe use genetic, RNA-sequencing, histology, flow cytometry, and imaging techniques to report the interaction between CCM endothelium, astrocytes, leukocytes, microglia/macrophages, neutrophils (CCM endothelium, astrocytes, leukocytes, microglia/macrophages, neutrophils interaction) during the pathogenesis of CCMs in the brain tissue.ResultsExpression profile of astrocytes in adult mouse brains using translated mRNAs obtained from the purification of EGFP (enhanced green fluorescent protein)-tagged ribosomes (Aldh1l1-EGFP/Rpl10a) in the presence or absence of CCM lesions (Slco1c1-iCreERT2;Pdcd10fl/fl; Pdcd10BECKO) identifies a novel gene signature for neuroinflammatory astrocytes. CCM-induced reactive astrocytes have a neuroinflammatory capacity by expressing genes involved in angiogenesis, chemotaxis, hypoxia signaling, and inflammation. RNA-sequencing analysis on RNA isolated from brain endothelial cells in chronic Pdcd10BECKO mice (CCM endothelium), identified crucial genes involved in recruiting inflammatory cells and thrombus formation through chemotaxis and coagulation pathways. In addition, CCM endothelium was associated with increased expression of Nlrp3 and Il1b. Pharmacological inhibition of NLRP3 (NOD [nucleotide-binding oligomerization domain]-' LRR [leucine-rich repeat]- and pyrin domain-containing protein 3) significantly decreased inflammasome activity as assessed by quantification of a fluorescent indicator of caspase-1 activity (FAM-FLICA [carboxyfluorescein-fluorochrome-labeled inhibitors of caspases] caspase-1) in brain endothelial cells from Pdcd10BECKO in chronic stage. Importantly, our results support the hypothesis of the crosstalk between astrocytes and CCM endothelium that can trigger recruitment of inflammatory cells arising from brain parenchyma (microglia) and the peripheral immune system (leukocytes) into mature active CCM lesions that propagate lesion growth, immunothrombosis, and bleedings. Unexpectedly, partial or total loss of brain endothelial NF-κB (nuclear factor κB) activity (using Ikkbfl/fl mice) in chronic Pdcd10BECKO mice does not prevent lesion genesis or neuroinflammation. Instead, this resulted in a trend increase in the number of lesions and immunothrombosis, suggesting that therapeutic approaches designed to target inflammation through endothelial NF-κB inhibition may contribute to detrimental side effects.ConclusionsOur study reveals previously unknown links between neuroinflammatory astrocytes and inflamed CCM endothelium as contributors that trigger leukocyte recruitment and precipitate immunothrombosis in CCM lesions. However, therapeutic approaches targeting brain endothelial NF-κB activity may contribute to detrimental side effects
The effect of epac activation on human coronary artery endothelial cells
The endothelial barrier is essential for vascular function, and its disruption may play a major role in the development of cardiovascular diseases. An increase in cAMP levels tightens the endothelial barrier by enhancing junction formation, and this is, in part, mediated by activation of exchange protein directly activated by cAMP (Epac). Vascular endothelial cells express vascular endothelial cadherin (VE-cadherin), and connexins 37, 40 and 43. Re-distribution of VE-cadherin in human coronary artery endothelial cells (HCAECs), induced by EGTA treatment or blocking VE-cadherin-VE-cadherin interactions through pre-treatment with anti-VE-cadherin primary antibody, triggered a subsequent re-distribution of Cx37 that was reversible. Epac activation with the Epac-selective agonist 8-pCPT induced a re-distribution of VE-cadherin and connexin 37 to sites of cell-cell contact in HCAECs, increasing the co-localisation of these two proteins, as detected by immunocytochemistry. This increased co-localisation was completely blocked by Epac1 siRNA. To test whether the re-distribution of Cx37 induced by Epac activation resulted in the formation of new functional gap junction channels, Lucifer yellow dye transfer was examined in HCAECs under varying conditions. Epac activation in HCAECs enhanced gap junction intercellular communication (GJIC), and this increase was completely blocked by the Epac inhibitor HJC0197. 8-pCPT addition was also shown to induce a transient increase in the intracellular calcium concentration in HCAECs, as detected with the calcium indicator Fluo-4. This calcium transient was independent of protein kinase A (PKA), and occurred in the absence of extracellular calcium, but was inhibited by the presence of the endoplasmic reticulum calcium ATPase inhibitor cyclopiazonic acid (CPA), indicating that Epac primarily mediated the increase, and that intracellular stores were the predominant source of calcium. Furthermore, the calcium transient induced by 8-pCPT was considerably reduced by the Epac inhibitor ESI-09 and completely inhibited by the Epac inhibitor HJC0197. Together, these data suggest that Epac strengthens the endothelial barrier through re-distribution of VE-cadherin and Cx37. In addition, GJIC is enhanced by Epac activation and there is a rise in intracellular calcium, a second messenger that can be transferred to adjacent cells through GJIC. It may be possible, therefore, that these effects of Epac promote endothelial cell- smooth muscle cell communication and therefore Epac could play a role in the regulation of vascular tone
Clinical pathophysiology: the essentials
ПОСОБИЯФИЗИОЛОГИЯПАТОЛОГИЯМЕДИЦИНА КЛИНИЧЕСКАЯСЕРДЕЧНО-СОСУДИСТАЯ СИСТЕМА /ПАТОФИЗИОЛОГИЯДЫХАТЕЛЬНАЯ СИСТЕМА /ПАТОФИЗИОЛОГИЯЖЕЛУДОЧНО-КИШЕЧНЫЙ ТРАКТ /ПАТОФИЗИОЛОГИЯПЕЧЕНЬ /ПАТОФИЗИОЛОГИЯПОЧКИ /ПАТОФИЗИОЛОГИЯЭНДОКРИННАЯ СИСТЕМА /ПАТОФИЗИОЛОГИЯНЕРВНАЯ СИСТЕМА /ПАТОФИЗИОЛОГИЯПАТОФИЗИОЛОГИЯИНОСТРАННЫЕ СТУДЕНТЫВ издании рассматриваются вопросы патофизиологии заболеваний основных систем организма, а также обсуждаются патофизиологические основы диагностики, профилактики и лечения заболеваний человека
Ras-like small GTPases in platelet biology
In a human body, the behaviour of each cell is controlled by signals like hormones or growth factors. A cell receives such messages by means of specific receptors. These transmit the messages to other proteins in the cell and so on. Such cascades control for example the pattern of active genes or metabolic responses. In this way, proliferation, differentiation, growth and cell death are tightly regulated. The complex of biochemical reactions that occurs in the cell upon signals is called signal transduction.
Members of the Ras-like small GTPases protein family function as molecular switches in signalling pathways. Activating signals induce the GTP-bound form of Ras proteins. In this conformation these proteins pass the signals on to other proteins. Hydrolysis of GTP returns them into the inactive, GDP-bound version and signalling is terminated. Certain mutations in the prototype family member Ras are known which render the protein continuously active. This contributes to oncogenic transformation. The function of the family members Rap1 and Ral is largely unknown. However, they are abundant and rapidly activated upon a variety of signals in platelets. This suggests these GTPases participate in signalling that controls platelet functions. Upon activation, platelets change their shape and concomitantly start sticking to each other (adhesion) by activation of adhesion molecules (integrins). This results in the formation of a blood clot which stops bleeding. Activation under inproper conditions may result in thrombosis, heart or brain infarct. Knowledge and insight with respect to signal transduction is thus required to understand platelet behaviour (and cells in general) under normal and pathological circumstances.
In this thesis, the focus has been on how Ras-like small GTPases are involved in signalling pathways as they occur in platelets. In chapter 1 the Ras proteins and their behaviour and function in blood platelets are discussed. In chapter 2 the calcium-induced Ras-independent Ral activation mechanism in platelets is studied. This research describes the identification and characterization of the Ral-specific guanine nucleotide exchange factor RalGEF2. In chapter 3, new insight is provided on the role of Rap1 in integrin-mediated cell adhesion. Rap1 is required for ?1 and ?2 integrin-mediated adhesion induced by Mn2+- or integrin activating antibodies. Treatment with Mn2+ or the activating antibodies did not induce Rap1 activation. Rap1 may fulfil a facilitating function. In chapter 4 the proposed Rap1 function in cAMP-induced PKA-independent elevation of the intracellular calcium concentration is investigated in megakaryocytes. Also in platelets Rap1 has been connected with the regulation of intracellular calcium concentration. However, no evidence was found for the involvement of Rap1 in this process. The experiments described in chapter 5 show Rap1 activation in a diversity of human blood cell lines representing megakaryocytes, monocytes, B and T lymphocytes as the consequence of shear stress. Moreover, Rap1 activity was required for ?IIb?3-mediated adhesion of human megakaryocytes. These findings suggest a connection between shear stress, Rap1 and integrin regulation, but also link Rap1 with the most important platelet integrin. Proper control of this integrin is inevitable for the correct functioning of blood platelets. This connects Rap1 with one of the most elementary processes in platelet activation
Identification of Novel Molecular Pathways Involved in Angiogenesis
In cancer and cardiovascular diseases (CVD), abnormal development of blood vessels plays a major role in disease progression. In case of cancer, abnormal angiogenesis promote tumour expansion and metastasis, which are characterized by formation of an abundant chaotic capillary network. In case of CVD, abnormal angiogenesis is characterised by obstruction of blood vessels, which results in hypoxia leading to cell apoptosis and necrosis in e.g. cardiac tissue. Many pathways of angiogenesis are still poorly understood and should be explored using gene function studies. In this thesis, we aim to identify novel molecular pathways involved in angiogenesis, which might shed light on failing gene therapy and offer novel perspectives on pro- and anti-angiogenic therapies.
To identify new genetic factors involved in angiogenesis, a genome-wide microarray screen was performed on the transcriptome of murine embryo`s. A list of genes that were significantly upregulated in FLK-positive cells was validated using whole-mount in situ hybridization in zebrafish larvae. Genes with a corresponding expression pattern in the vasculature were selected and silenced in an in vivo model of angiogenesis, in developing zebrafish larvae and in a mural retina model. In vitro knockdown assays were also performed in 3D-collagen based co-culture of EC and pericytes to assess blood vessel formation. In this thesis CMTM3, CMTM4, CGNL1, THSD1, and CECR1 were selected as candidate genes and their molecular pathways were investigated. For the first time to our knowledge we concluded that their properties in ECs are involved in the angiogenesis process. We also investigated the angiogenic property of the nucleotide Up4A as it is synthesised in response to VEGFR2 stimulation during stress conditions, such as hypoxia or in cancer conditions.
In conclusion, our fundamental findings of novel pathways involved in angiogenesis hold promising therapeutic perspectives to understand and treat vascular diseases