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The capillary-venous pathology cerebral cavernous malformation (CCM) is caused by loss of CCM1/Krev interaction trapped protein 1 (KRIT1), CCM2/MGC4607, or CCM3/PDCD10 in some endothelial cells. Mutations of CCM genes within the brain vasculature can lead to recurrent cerebral hemorrhages. Pharmacological treatment options are urgently needed when lesions are located in deeply-seated and in-operable regions of the central nervous system. Previous pharmacological suppression screens in disease models of CCM led to the discovery that treatment with retinoic acid improved CCM phenotypes. This finding raised a need to investigate the involvement of retinoic acid in CCM and test whether it has a curative effect in preclinical mouse models. Here, we show that components of the retinoic acid synthesis and degradation pathway are transcriptionally misregulated across disease models of CCM. We complemented this analysis by pharmacologically modifying retinoic acid levels in zebrafish and human endothelial cell models of CCM, and in acute and chronic mouse models of CCM. Our pharmacological intervention studies in CCM2-depleted human umbilical vein endothelial cells (HUVECs) and krit1 mutant zebrafish showed positive effects when retinoic acid levels were increased. However, therapeutic approaches to prevent the development of vascular lesions in adult chronic murine models of CCM were drug regiment-sensitive, possibly due to adverse developmental effects of this hormone. A treatment with high doses of retinoic acid even worsened CCM lesions in an adult chronic murine model of CCM. This study provides evidence that retinoic acid signaling is impaired in the CCM pathophysiology and suggests that modification of retinoic acid levels can alleviate CCM phenotypes., (© 2023. The Author(s).)

Background and Purpose- Cerebral cavernous malformations (CCMs) are vascular malformations of the brain that lead to cerebral hemorrhages. A pharmacological treatment is needed especially for patients with nonoperable deep-seated lesions. We and others obtained CCM mouse models that were useful for mechanistic studies and rapid trials testing the preventive effects of candidate drugs. The shortened lifespan of acute mouse models hampered evaluation of compounds that would not only prevent lesion appearance but also cure preexisting lesions. Indirubin-3'-monoxime previously demonstrated its efficacy to reverse the cardiac phenotype of ccm2 m201 zebrafish mutants and to prevent lesion development in an acute CCM2 mouse model. In the present article, we developed and characterized a novel chronic CCM2 mouse model and evaluated the curative therapeutic effect of indirubin-3'-monoxime after CCM lesion development. Methods- The chronic mouse model was obtained by a postnatal induction of brain-endothelial-cell-specific ablation of the Ccm2 gene using the inducible Slco1c1 -CreER T2 mouse line. Results- We obtained a fully penetrant novel CCM chronic mouse model without any obvious off-target phenotypes and compatible with long-term survival. By 3 months of age, CCM lesions ranging in size from small isolated lesions to multiple caverns developed throughout the brain. Lesion burden was quantified in animals from 1 week to 5 months of age. Clear signs of intracerebral hemorrhages were noticed in brain-endothelial-cell-specific ablation of the Ccm2 gene. In contrast with its preventive effect in the acute CCM2 mouse model, a 20 mg/kg indirubin-3'-monoxime treatment for 3 weeks in 3-month old animals neither had any beneficial effect on the lesion burden nor alleviated cerebral hemorrhages. Conclusions- The brain-endothelial-cell-specific ablation of the Ccm2 gene chronic model is a strongly improved disease model for the CCM community whose challenge today is to decipher which candidate drugs might have a curative effect on patients' preexisting lesions. Visual Overview- An online visual overview is available for this article.

Cerebral cavernous malformations (CCMs) are vascular lesions in the central nervous system causing strokes and seizures which currently can only be treated through neurosurgery. The disease arises through changes in the regulatory networks of endothelial cells that must be comprehensively understood to develop alternative, non-invasive pharmacological therapies. Here, we present the results of several unbiased small-molecule suppression screens in which we applied a total of 5,268 unique substances to CCM mutant worm, zebrafish, mouse, or human endothelial cells. We used a systems biology-based target prediction tool to integrate the results with the whole-transcriptome profile of zebrafish CCM2 mutants, revealing signaling pathways relevant to the disease and potential targets for small-molecule-based therapies. We found indirubin-3-monoxime to alleviate the lesion burden in murine preclinical models of CCM2 and CCM3 and suppress the loss-of-CCM phenotypes in human endothelial cells. Our multi-organism-based approach reveals new components of the CCM regulatory network and foreshadows novel small-molecule-based therapeutic applications for suppressing this devastating disease in patients., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)

Endothelial integrity relies on a mechanical crosstalk between intercellular and cell-matrix interactions. This crosstalk is compromised in hemorrhagic vascular lesions of patients carrying loss-of-function mutations in cerebral cavernous malformation (CCM) genes. RhoA/ROCK-dependent cytoskeletal remodeling is central to the disease, as it causes unbalanced cell adhesion towards increased cell-extracellular matrix adhesions and destabilized cell-cell junctions. This study reveals that CCM proteins directly orchestrate ROCK1 and ROCK2 complementary roles on the mechanics of the endothelium. CCM proteins act as a scaffold, promoting ROCK2 interactions with VE-cadherin and limiting ROCK1 kinase activity. Loss of CCM1 (also known as KRIT1) produces excessive ROCK1-dependent actin stress fibers and destabilizes intercellular junctions. Silencing of ROCK1 but not ROCK2 restores the adhesive and mechanical homeostasis of CCM1 and CCM2-depleted endothelial monolayers, and rescues the cardiovascular defects of ccm1 mutant zebrafish embryos. Conversely, knocking down Rock2 but not Rock1 in wild-type zebrafish embryos generates defects reminiscent of the ccm1 mutant phenotypes. Our study uncovers the role of the CCM1-CCM2 complex in controlling ROCK1 and ROCK2 to preserve endothelial integrity and drive heart morphogenesis. Moreover, it solely identifies the ROCK1 isoform as a potential therapeutic target for the CCM disease., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Motivation: Most computational approaches for the analysis of omics data in the context of interaction networks have very long running times, provide single or partial, often heuristic, solutions and/or contain user-tuneable parameters., Results: We introduce local enrichment analysis (LEAN) for the identification of dysregulated subnetworks from genome-wide omics datasets. By substituting the common subnetwork model with a simpler local subnetwork model, LEAN allows exact, parameter-free, efficient and exhaustive identification of local subnetworks that are statistically dysregulated, and directly implicates single genes for follow-up experiments., Evaluation on simulated and biological data suggests that LEAN generally detects dysregulated subnetworks better, and reflects biological similarity between experiments more clearly than standard approaches. A strong signal for the local subnetwork around Von Willebrand Factor (VWF), a gene which showed no change on the mRNA level, was identified by LEAN in transcriptome data in the context of the genetic disease Cerebral Cavernous Malformations (CCM). This signal was experimentally found to correspond to an unexpected strong cellular effect on the VWF protein. LEAN can be used to pinpoint statistically significant local subnetworks in any genome-scale dataset., Availability and Implementation: The R-package LEANR implementing LEAN is supplied as supplementary material and available on CRAN ( https://cran.r-project.org )., Contacts: benno@pasteur.fr or tournier-lasserve@univ-paris-diderot.fr., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2016. Published by Oxford University Press.)

Cerebral cavernous malformation (CCM) is a disease of the central nervous system causing hemorrhage-prone multiple lumen vascular malformations and very severe neurological consequences. At present, the only recommended treatment of CCM is surgical. Because surgery is often not applicable, pharmacological treatment would be highly desirable. We describe here a murine model of the disease that develops after endothelial-cell-selective ablation of the CCM3 gene. We report an early, cell-autonomous, Wnt-receptor-independent stimulation of β-catenin transcription activity in CCM3-deficient endothelial cells both in vitro and in vivo and a triggering of a β-catenin-driven transcription program that leads to endothelial-to-mesenchymal transition. TGF-β/BMP signaling is then required for the progression of the disease. We also found that the anti-inflammatory drugs sulindac sulfide and sulindac sulfone, which attenuate β-catenin transcription activity, reduce vascular malformations in endothelial CCM3-deficient mice. This study opens previously unidentified perspectives for an effective pharmacological therapy of intracranial vascular cavernomas.

Background and Purpose: Cerebral cavernous malformation (CCM) is a neurovascular dysplasia characterized by conglomerates of enlarged endothelial channels in the central nervous system, which are almost devoid of pericytes or smooth muscle cells. This disease is caused by loss-of-function mutations in CCM1, CCM2, or CCM3 genes in endothelial cells, making blood vessels highly susceptible to angiogenic stimuli. CCM1- and CCM3-silenced endothelial cells have a reduced expression of the Notch ligand Delta-like 4 (DLL4) resulting in impaired Notch signaling and irregular sprouting angiogenesis. This study aimed to address if DLL4, which is exclusively expressed on endothelial cells, may influence interactions of endothelial cells with pericytes, which express Notch3 as the predominant Notch receptor., Methods: Genetic manipulation of primary human endothelial cells and brain pericytes. Transgenic mouse models were also used., Results: Endothelial cell-specific ablation of Ccm1 and Ccm2 in different mouse models led to the formation of CCM-like lesions, which were poorly covered by periendothelial cells. CCM1 silencing in endothelial cells caused decreased Notch3 activity in cocultured pericytes. DLL4 proteins stimulated Notch3 receptors on human brain pericytes. Active Notch3 induced expression of PDGFRB2, N-Cadherin, HBEGF, TGFB1, NG2, and S1P genes. Notch3 signaling in pericytes enhanced the adhesion strength of pericytes to endothelial cells, limited their migratory and invasive behavior, and enhanced their antiangiogenic function. Pericytes silenced for Notch3 expression were more motile and could not efficiently repress angiogenesis., Conclusions: The data suggest that Notch signaling in pericytes is important to maintain the quiescent vascular phenotype. Deregulated Notch signaling may, therefore, contribute to the pathogenesis of CCM., (© 2015 American Heart Association, Inc.)

Mechanotransduction pathways are activated in response to biophysical stimuli during the development or homeostasis of organs and tissues. In zebrafish, the blood-flow-sensitive transcription factor Klf2a promotes VEGF-dependent angiogenesis. However, the means by which the Klf2a mechanotransduction pathway is regulated to prevent continuous angiogenesis remain unknown. Here we report that the upregulation of klf2 mRNA causes enhanced egfl7 expression and angiogenesis signaling, which underlies cardiovascular defects associated with the loss of cerebral cavernous malformation (CCM) proteins in the zebrafish embryo. Using CCM-protein-depleted human umbilical vein endothelial cells, we show that the misexpression of KLF2 mRNA requires the extracellular matrix-binding receptor β1 integrin and occurs in the absence of blood flow. Downregulation of β1 integrin rescues ccm mutant cardiovascular malformations in zebrafish. Our work reveals a β1 integrin-Klf2-Egfl7-signaling pathway that is tightly regulated by CCM proteins. This regulation prevents angiogenic overgrowth and ensures the quiescence of endothelial cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Moyamoya is a cerebrovascular condition characterized by a progressive stenosis of the terminal part of the internal carotid arteries (ICAs) and the compensatory development of abnormal "moyamoya" vessels. The pathophysiological mechanisms of this condition, which leads to ischemic and hemorrhagic stroke, remain unknown. It can occur as an isolated cerebral angiopathy (so-called moyamoya disease) or in association with various conditions (moyamoya syndromes). Here, we describe an autosomal-recessive disease leading to severe moyamoya and early-onset achalasia in three unrelated families. This syndrome is associated in all three families with homozygous mutations in GUCY1A3, which encodes the α1 subunit of soluble guanylate cyclase (sGC), the major receptor for nitric oxide (NO). Platelet analysis showed a complete loss of the soluble α1β1 guanylate cyclase and showed an unexpected stimulatory role of sGC within platelets. The NO-sGC-cGMP pathway is a major pathway controlling vascular smooth-muscle relaxation, vascular tone, and vascular remodeling. Our data suggest that alterations of this pathway might lead to an abnormal vascular-remodeling process in sensitive vascular areas such as ICA bifurcations. These data provide treatment options for affected individuals and strongly suggest that investigation of GUCY1A3 and other members of the NO-sGC-cGMP pathway is warranted in both isolated early-onset achalasia and nonsyndromic moyamoya., (Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The endothelial CCM complex regulates blood vessel stability and permeability. Loss-of-function mutations in CCM genes are responsible for human cerebral cavernous malformations (CCMs), which are characterized by clusters of hemorrhagic dilated capillaries composed of endothelium lacking mural cells and altered sub-endothelial extracellular matrix (ECM). Association of the CCM1/2 complex with ICAP-1, an inhibitor of β1 integrin, prompted us to investigate whether the CCM complex interferes with integrin signaling. We demonstrate that CCM1/2 loss resulted in ICAP-1 destabilization, which increased β1 integrin activation and led to increased RhoA-dependent contractility. The resulting abnormal distribution of forces led to aberrant ECM remodeling around lesions of CCM1- and CCM2-deficient mice. ICAP-1-deficient vessels displayed similar defects. We demonstrate that a positive feedback loop between the aberrant ECM and internal cellular tension led to decreased endothelial barrier function. Our data support that up-regulation of β1 integrin activation participates in the progression of CCM lesions by destabilizing intercellular junctions through increased cell contractility and aberrant ECM remodeling.

Little is known about the molecular mechanisms that regulate the organization of vascular lumen. In this paper we show that lumen formation correlates with endothelial polarization. Adherens junctions (AJs) and VE-cadherin (VEC, encoded by CDH5) are required for endothelial apicobasal polarity in vitro and during embryonic development. Silencing of CDH5 gene expression leads to abrogation of endothelial polarity accompanied by strong alterations in lumenal structure. VEC co-distributes with members of the Par polarity complex (Par3 and PKCzeta) and is needed for activation of PKCzeta. CCM1 is encoded by the CCM1 gene, which is mutated in 60% of patients affected by cerebral cavernous malformation (CCM). The protein interacts with VEC and directs AJ organization and AJ association with the polarity complex, both in cell-culture models and in human CCM1 lesions. Both VEC and CCM1 control Rap1 concentration at cell-cell junctions. We propose that VEC, CCM1 and Rap1 form a signaling complex. In the absence of any of these proteins, AJs are dismantled, cell polarity is lost and vascular lumenal structure is severely altered.

Cerebral cavernous malformations (CCM) are vascular lesions which can occur as a sporadic (80% of the cases) or familial autosomal dominant form (20%). Three CCM genes have been identified: CCM1/KRIT1, CCM2/MGC4607 and CCM3/PDCD10. Almost 80% of CCM patients affected with a genetic form of the disease harbor a heterozygous germline mutation in one of these three genes. Recent work has shown that a two-hit mechanism is involved in CCM pathogenesis which is caused by a complete loss of any of the three CCM proteins within endothelial cells lining the cavernous capillary cavities. These data were an important step towards the elucidation of the mechanisms of this condition.

Cerebral cavernous malformations (CCM) are vascular malformations of the brain that lead to cerebral hemorrhages. In 20% of CCM patients, this results from an autosomal dominant condition caused by loss-of-function mutations in one of the three CCM genes. High expression levels of the CCM genes in the neuroepithelium indicate that CCM lesions might be caused by a loss of function of these genes in neural cells rather than in vascular cells. However, their in vivo function, particularly during cerebral angiogenesis, is totally unknown. We developed mice with constitutive and tissue-specific CCM2 deletions to investigate CCM2 function in vivo. Constitutive deletion of CCM2 leads to early embryonic death. Deletion of CCM2 from neuroglial precursor cells does not lead to cerebrovascular defects, whereas CCM2 is required in endothelial cells for proper vascular development. Deletion of CCM2 from endothelial cells severely affects angiogenesis, leading to morphogenic defects in the major arterial and venous blood vessels and in the heart, and results in embryonic lethality at mid-gestation. These findings establish the essential role of endothelial CCM2 for proper vascular development and strongly suggest that the endothelial cell is the primary target in the cascade of events leading from CCM2 mutations to CCM cerebrovascular lesions.

Background: Chemokines are well-established to function in the recruitment of leukocytes into allografts in the course of rejection. Moreover, some studies have indicated that there are organ-specific differences in chemokine function, but the mechanism accounting for this difference is not known., Methods: Fully major histocompatibility complex-mismatched vascularized cardiac transplants or skin transplants were performed using BALB/c (H-2d), C57BL/6 (H-2b), MCP-1-/- (H-2b) and CXCR3-/- (H-2b) mice as donors or recipients. Also, skin grafts (H-2b) were placed onto SCID mice (H-2d) that received BALB/c splenocytes (H-2d) by adoptive transfer either at the time of transplantation, or after a period of 28 days., Results: Cardiac allografts in MCP-1-/- recipients survived significantly longer (P<0.0005) than wild-type (WT) controls. However, there was no prolongation of survival when MCP-1-/- grafts were used a donors in WT mice. In contrast, the absence of donor but not recipient MCP-1 prolonged skin allograft survival. WT donor cardiac grafts in CXCR3-/- recipients had a modest prolongation of survival (P<0.0005), whereas CXCR3-/- donor cardiac grafts in WT recipients were rejected similar to controls. Also, while recipient CXCR3 had no effect on the rejection of skin, CXCR3-/- donor skin grafts survived significantly longer than WT controls. This survival advantage was lost when vascularized CXCR3-/- skin grafts were used as donors in the SCID model of rejection., Conclusion: Recipient derived MCP-1 and CXCR3 are functional in the rejection of vascularized, but not nonvascularized, allografts. In contrast, donor-derived MCP-1 and CXCR3 are functional in nonvascularized, but not vascularized grafts.

Lnk, with APS and SH2-B (Src homology 2-B), belongs to a family of SH2-containing proteins with potential adaptor functions. Lnk regulates growth factor and cytokine receptor-mediated pathways implicated in lymphoid, myeloid, and platelet homeostasis. We have previously shown that Lnk is expressed and up-regulated in vascular endothelial cells (ECs) in response to tumor necrosis factor-alpha (TNFalpha). In this study, we have shown that, in ECs, Lnk down-regulates the expression, at both mRNA and protein levels, of the proinflammatory molecules VCAM-1 and E-selectin induced by TNFalpha. Mechanistically, our data indicated that, in response to TNFalpha, NFkappaB/p65 phosphorylation and translocation as well as IkappaBalpha phosphorylation and degradation were unchanged, suggesting that Lnk does not modulate NFkappaB activity. However, Lnk activates phosphatidylinositol 3-kinase (PI3K) as reflected by Akt phosphorylation. Our results identify endothelial nitric-oxide synthase as a downstream target of Lnk-mediated activation of the PI3K/Akt pathway and HO-1 as a new substrate of Akt. We found that sustained Lnk-mediated activation of PI3K in TNFalpha-activated ECs correlated with the inhibition of ERK1/2 phosphorylation, whereas phosphorylation of p38 and c-Jun NH(2)-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) was unchanged. ERK1/2 inhibition decreases VCAM-1 expression in TNFalpha-treated ECs. Collectively, our results identify the adaptor Lnk as a negative regulator in the TNFalpha-signaling pathway mediating ERK inhibition and suggest a role for Lnk in the interplay between PI3K and ERK triggered by TNFalpha in ECs.

Vascular endothelial growth factor (VEGF), an angiogenesis factor, has recently been found to have potent proinflammatory properties in vivo. However, the mechanism by which it mediates inflammation is poorly understood. In this study, we have evaluated the function of VEGF on the induced expression and function of the T cell chemoattractant chemokine IFN-gamma-inducible protein of 10 kDa (IP-10). In vitro, we find that VEGF augments the effect of IFN-gamma on the induction of IP-10 mRNA and protein expression in endothelial cells. Moreover, we show that VEGF and IFN-gamma regulate the activation of the IP-10 promoter, and that the kinases PI3K, phosphoinositide-dependent kinase 1, and Akt act as intermediary signaling molecules for cytokine-inducible IP-10 transcriptional activation in endothelial cells. To examine whether VEGF is functional for IP-10 expression in vivo, Chinese hamster ovary cells that were designed to secrete VEGF were injected s.c. into the skin of nude mice and were found to mediate a time-dependent increase in IP-10 mRNA. This response was reduced in animals treated systemically with the PI3K inhibitor wortmannin. When the Chinese hamster ovary cells expressing VEGF plasmid were injected s.c. into C57BL/6 wild-type or CXCR3-/- mice, they elicited an inflammatory reaction in wild-type but not in CXCR3-/- mice. Collectively, these findings indicate that VEGF-induced augmentation of IP-10 expression is a major mechanism underlying its proinflammatory function.

Lnk is an adaptator protein involved in B lymphocytes and platelet differentiation and in T lymphocyte activation. We previously reported on Lnk expression and regulation in endothelial cells (ECs) upon activation. In the present study, the involvement of Lnk in the tumor necrosis factor alpha (TNFalpha) pathway was investigated in vitro through Lnk overexpression in primary cultures of human endothelial cells. Using a recombinant adenovirus encoding human Lnk, we first demonstrated that Lnk overexpression does not induce vascular cell adhesion molecule-1 (VCAM-1) suggesting that Lnk does not promote ECs activation. However, Lnk overexpression significantly reduced TNFalpha-mediated expression of VCAM-1 (at mRNA and protein levels) in activated EC as compared with controls. Western blot analysis showed that Lnk overexpression in HUVEC was associated with phosphorylation of Akt kinase (at Ser 473) with no effect on IkappaBalpha, the specific inhibitor of NFkappaB, indicating that Lnk promotes activation of the phosphatidylinositol 3-kinase (PI3-kinase) pathway in ECs. Altogether, these results suggest that, in ECs, Lnk may participate to a regulatory pathway involving the PI3-kinase and modulating the inflammatory response.

Control of molecular targets and signaling pathways which improve endothelial cell survival may be an attractive concept for interfering with dysregulated vascular injury and remodeling, a key mechanism for transplant arteriosclerosis and chronic allograft rejection. In addition to inhibiting matrix metalloproteinase activity, it has been suggested by recent studies that the tissue inhibitor of metalloproteinase (TIMP)-1 may inhibit apoptosis in various cell types. The present work examines the possibility that TIMP-1 belongs to a protective pathway via antiapoptotic properties and investigates the signaling pathway mediated by TIMP-1 in human ECs. We demonstrate that exogenous, recombinant, TIMP-1 efficiently prevents apoptosis induced by TNFalpha in cycloheximide-sensitized ECs. The antiapoptotic effect of TIMP-1 was dose-dependent and a maximal effect of TIMP-1 (30% protection) was reached using 250 ng/mL of recombinant TIMP-1. We present evidence that TIMP-1 induces activation of PI3-kinase but not NFkappaB pathway in ECs. Our findings further indicate that TIMP-1-induced EC survival is mediated through activation of PI3-kinase pathway and the downstream phosphorylation of Akt kinase. Blocking the PI3-kinase pathway with wortmannin or LY294002 restores TNFalpha-mediated EC death. In conclusion, our findings suggest that TIMP-1, generated upon inflammation, acts as an antiapoptotic molecule that can prevent EC apoptosis through activation of the PI3-kinase and phosphorylation of the Akt kinase.

HLA class I ligation on graft endothelial cells (EC) has been shown to promote graft arteriosclerosis and chronic allograft nephropathy. This study investigated transcriptional and functional changes mediated by anti-HLA antibodies (Ab), developed by transplant recipient, on vascular renal EC. For mimicking interactions that occur between alloantibodies and graft endothelium, HLA-typed primary cultures of human EC were incubated in vitro in the presence of monomorphic or polymorphic anti-HLA class I Ab. Gene expression analysis identified the upregulation of several molecules involved in cell signaling and proliferation, including the GTP-binding protein RhoA. It was demonstrated further that HLA class I ligation on EC induced a rapid translocation of RhoA to the cell membrane associated with F-actin stress fiber formation and cytoskeleton reorganization. Western blot analysis showed that anti-HLA class I Ab induced, in addition to RhoA, the activation of phosphatidylinositol 3-kinase, reflected by the phosphorylation of Akt (Ser473) and GSK3beta (Ser9), in EC. C3 exoenzyme, an inhibitor of RhoA, inhibited RhoA translocation in response to HLA class I ligation and reduced phosphatidylinositol 3-kinase activation. EC proliferation and cell cycle progression, examined by 5,6-carboxyfluorescein diacetate succinimidyl ester staining, demonstrated that anti-HLA-induced EC proliferation was efficiently prevented by the 3-hydroxy-3-methylglutaryl CoA reductase inhibitor simvastatin (0.1 micromol/L) through inhibition of RhoA geranylgeranylation. Taken together, these findings support the conclusion that RhoA is a key mediator of signaling pathways that lead to cytoskeletal reorganization and EC proliferation in response to alloantibodies that bind to HLA class I and demonstrate the specific and potent inhibitory effect of simvastatin on allostimulated EC growth.

Besides virological and physiological concerns, the success of xenotransplantation (Xt) is still dependent on the prevention of delayed xenograft rejection (DXR). Although multifactorial, DXR is mainly due to xenonatural antibody (Ab) recognizing their xenogenic antigen (Ag) followed by complement activation. Despite the use of intensive treatments capable of inhibiting the humoral response, DXR can still not be avoided and always occurs within weeks following transplantation. Moreover, these latter treatments currently used in Xt could not be used clinically in humans because of their high risk of over-immunosuppressing the patients. Mitoxantrone (Mx) is a drug well known for its antiproliferative properties and is used clinically in oncology and in the treatment of relapsing multiple sclerosis. In models of arthritis in rats, it has been shown to be 10 to 20 times more powerful than cyclophosphamide (CyP) at blocking both inflammatory and B-cell responses. Because of its B-cell inhibitory capacity and considering the implication of the humoral response in xenograft rejection, we have compared Mx with CyP for its ability to block in vivo anti-pig immunization induced via subcutaneous injections of pig red blood cells into baboons. Neither drug was able to inhibit the anti-pig responses following the first and second immunizations, emphasizing the particularity of preformed Ab responses. However, the rise in Ab in the Mx treated animals was significantly delayed as compared with the non-treated as well as the CyP treated animals and was mainly because of a profound depletion of circulating B-cells. Mx displays an interesting antihumoral effect that we now intend to test in a pig kidney to baboon Xt model, with anticipated administration of the drug allowing an early B-cell depletion.

Background: A better understanding of inflammatory processes in endothelial cells (ECs) might reveal new ways of controlling inflammation and graft rejection. This study investigates EC genes regulated in response to human tumor necrosis factor (TNF)-alpha and xenogeneic natural antibodies (XNAs) that contribute to endothelial activation during transplantation., Methods: Gene expression between resting and activated ECs was investigated by RNA differential display reverse-transcriptase polymerase chain reaction and confirmed by reverse-Northern blot., Results: Forty-five cDNA fragments corresponding to genes up-regulated in activated ECs were identified. Our findings show that TNF-alpha-mediated EC activation was associated with increased levels of mRNA for the adaptor protein Lnk, the nuclear protein RED, and the initiation factor eIF4G. We further show that Lnk and eIF4G were also up-regulated in response to XNA binding to ECs., Conclusion: Our data suggest that TNF-alpha and XNAs could share common signaling pathways involving Lnk and eIF4G but may also drive specific transcriptional events.

Background: RNA differential display (DD) RT-PCR is a useful method to identify and clone differentially expressed genes. However, the rate of false positives and redundancy associated with this PCR-based method as well as laborious downstream screening steps constitute major limitations. Here we present DD RT-PCR and reverse northern (RN) protocols allowing rapid and acurate identification of genes upregulated in porcine endothelial cells (EC) in response to TNFalpha., Materials and Methods: The housekeeping gene beta-actin was used to investigate mispriming and to set up optimal conditions for DD-RT-PCR and RN. In this study DD was performed to compare resting and TNFalpha-activated ECs. Selection of DD-fragments was performed following 30-cycles of PCR using serial dilutions of template cDNA and regulation of 6 out of 17 candidates genes were first confirmed by semi-quantitative RN., Results: Using this protocol, 5 out of 6 DD-fragments were further confirmed to be upregulated by Northern blot, and 3 novel porcine cDNAs were cloned including the pro-apoptotic member of the Bcl-2 family, Noxa., Conclusion: In this study we demonstrate that the combination of DD-RT-PCR and RN, which efficiently reduces the number of false positive candidates derived from mispriming at the screening step, allows a rapid identification of differentially expressed genes.

Background: Effective improvement in xenograft survival is achieved using transplants from transgenic pigs expressing human complement (C) regulatory proteins, including decay-accelerating factor (DAF), CD59, and CD46 on endothelial cells (ECs). The aim of this study was to investigate whether human DAF expression in porcine ECs, as well as regulating C activation, can modify intercellular events through its interaction with its receptor, CD97, on human leukocytes., Methods: Cellular interactions between human leukocytes and porcine ECs were investigated in vitro using ECs from either wild-type or DAF-transgenic pigs. Static leukocyte adhesion and T cell activation assays were performed using porcine ECs as target or effector cells, respectively. The role of the DAF-CD97 interaction was investigated using specific blocking monoclonal antibodies (mAbs) against human DAF and its receptor, CD97, in adhesion assays., Results: Adhesion of U937 or Jurkat T cells, both expressing human DAF and CD97, was quantitatively similar for wild-type and transgenic-DAF-expressing pig ECs. Furthermore, blocking the CD97-DAF interaction did not inhibit xenogeneic leukocyte-endothelium adhesion, whereas blocking the very late antigen 4-vascular cell adhesion molecule-1 pathway reduced this adhesion by 50-80%. Furthermore, DAF and CD97 expression was not up-regulated during tumor necrosis factor-alpha- or lipopolysaccharide-mediated EC activation, unlike the adhesion molecules E-selectin, vascular cell adhesion molecule-1, and intracellular adhesion molecule (ICAM)-1., Conclusion: We found that high levels of human DAF expressed on ECs abrogates C-mediated cell damage but did not affect the in vitro adhesive properties or antigen-presenting cell function of genetically modified porcine ECs.

Endothelial cell (EC) interaction with antigraft antibodies (Abs) mediates EC injury and activation involved in vascular graft rejection. The aim of this study was to identify EC genes regulated in response to antigraft Ab binding that contribute to the endothelium alterations implicated in graft rejection or survival. By means of RNA differential display, 13 cDNA fragments corresponding to genes differentially expressed in ECs incubated with antigraft Abs were identified. Among these cDNAs were found the tissue inhibitor of metalloproteinase-1 (TIMP-1) and a desintegrin and metalloproteinase (ADAM-10). We demonstrated that TIMP-1 and ADAM-10 mRNA and protein expression was rapidly upregulated in ECs in response to antigraft Ab binding. Our data showed that TIMP-1 was upregulated in response to human IgG but not IgM and anti-galactosyl (Gal) alpha1-3Gal human xenogeneic Abs. In contrast, upregulation of ADAM-10 in ECs was shown to be mostly mediated by anti-Galalpha1-3Gal IgM Abs. Specific effects of human IgG and IgM xenogeneic Abs on endothelial transcripts indicate that different isotypes and specificities of Abs may mediate different EC changes. Our results suggest that interaction of ECs with antigraft Abs, according to their specificity, selectively induces synthesis and release of metalloproteinases and inhibitors, controlling proteolytic processes and immunological events that respectively contribute to graft rejection or survival.

Background: Cyclosporine (CsA) is currently given to recipients of vascularized xenografts as part of the immunosuppressive regimen required to prevent the hyperacute rejection phase. The effects of CsA on non-lymphoid immune cells, such as endothelial cells (ECs), have not been well characterized and sometimes seem contradictory, because both protective and adverse effects have been reported. In the present study, we investigated in vitro whether CsA could alter the antigenicity of activated porcine aortic endothelial cells (PAECs) by reducing class I and class II MHC antigen expression., Methods: The effect of CsA on MHC antigen expression during tumor necrosis factor (TNF)-alpha- or lymphocyte-mediated PAEC activation was evaluated in vitro by flow cytometry and correlated to the ability of porcine ECs to promote human T lymphocyte proliferation. The effect of CsA on class II MHC antigen mRNA expression was also analyzed and related to class II transcriptional activator (CIITA) mRNA expression., Results: Flow cytometry analysis showed that TNF-alpha-mediated induction of class II MHC antigen expression on PAECs was completely inhibited by CsA, whereas expression of class I MHC was reduced by 50%. The inhibition was dose dependent (at drug concentrations ranging from 2.5 microg/ml to 20.0 microg/ml) and was consistently observed at all time points (24-72 hr) during the activation period. Decreased MHC antigen expression dramatically reduced the ability of PAECs to further promote human T-cell proliferation. Similar levels of inhibition were achieved using an anti-porcine class II MHC blocking monoclonal antibody. Pretreatment of PAECs with CsA for 4 hr before coculture with human peripheral blood leukocytes efficiently blocked the induction on PAECs of E-selectin and class II MHC antigens and inhibited overexpression of class I antigens. Semiquantitative reverse transcriptase-polymerase chain reaction experiments showed that CsA markedly reduced the steady-state level of porcine class II (SLA-DRA and SLA-DQA) mRNA at 16 hr, compared with PAECs stimulated with TNF-alpha alone. The reduced level of class II MHC mRNA was associated with a lack of CIITA expression at this time point, suggesting that CsA could alter transcription or promote the rapid decay of CIITA mRNA., Conclusion: Our study indicates that CsA could play a role in preventing porcine MHC antigens being directly presented to human T lymphocytes by xenogeneic ECs.