Gene expression study in positron emission tomography-positive abdominal aortic aneurysms identifies CCL18 as a potential biomarker for rupture risk.

Rupture of abdominal aortic aneurysm (AAA) is a cause of significant mortality and morbidity in aging populations. Uptake of 18-fluorodeoxyglucose (FDG) detected by positron emission tomography (PET) is observed in the wall of 12% of AAA (A+), with most of them being symptomatic. We previously showed that the metabolically active areas displayed adventitial inflammation, medial degeneration and molecular alterations prefacing wall rupture. The aim of this study was to identify new factors predictive of rupture. Transcriptomic analyses were performed in the media and adventitia layers from three types of samples: AAA with-out FDG uptake (A0) and with FDG uptake (A+), both at the positive spot (A+(Pos)) and at a paired distant negative site (A+(Neg)) of the same aneurysm. Follow-up studies included reverse-transcriptase-polymerase chain reaction (RT-PCR), immunohistochemical staining and enzyme-linked immunosorbent assay (ELISA). A large number of genes, including matrix metalloproteinases, collagens and cytokines as well as genes involved in osteochondral development, were differentially expressed in the A+(Pos) compared with A+(Neg). Moreover, a series of genes (notably CCL18) was differentially expressed both in the A+(Neg) and A+(Pos) compared with the A0. A significant increase of CCL18 was also found at the protein level in the aortic wall and in peripheral blood of A+ patients compared with A0. In conclusion, new factors, including CCL18, involved in the progression of AAA and, potentially, in their rupture were identified by a genome-wide analysis of PET-positive and -negative human aortic tissue samples. Further work is needed to study their role in AAA destabilization and weakening.

Effects of platelet-rich plasma (PRP) on the healing of Achilles tendons of rats.

Platelet-rich plasma (PRP) contains growth factors involved in the tissular healing process. The aim of the study was to determine if an injection of PRP could improve the healing of sectioned Achilles tendons of rats. After surgery, rats received an injection of PRP (n = 60) or a physiological solution (n = 60) in situ. After 5, 15, and 30 days, 20 rats of both groups were euthanized and 15 collected tendons were submitted to a biomechanical test using cryo-jaws before performing transcriptomic analyses. Histological and biochemical analyses were performed on the five remaining tendons in each group. Tendons in the PRP group were more resistant to rupture at 15 and 30 days. The mechanical stress was significantly increased in tendons of the PRP group at day 30. Histological analysis showed a precocious deposition of fibrillar collagen at day 5 confirmed by a biochemical measurement. The expression of tenomodulin was significantly higher at day 5. The messenger RNA levels of type III collagen, matrix metalloproteinases 2, 3, and 9, were similar in the two groups at all time points, whereas type I collagen was significantly increased at day 30 in the PRP group. In conclusion, an injection of PRP in sectioned rat Achilles tendon influences the early phase of tendon healing and results in an ultimately stronger mechanical resistance.

Human bone marrow adipocytes block granulopoiesis through neuropilin-1-induced granulocyte colony-stimulating factor inhibition.

Adipocytes are part of hematopoietic microenvironment, even though up to now in humans, their role in hematopoiesis is still questioned. We have previously shown that accumulation of fat cells in femoral bone marrow (BM) coincides with increased expression of neuropilin-1 (NP-1), while it is weakly expressed in hematopoietic iliac crest BM. Starting from this observation, we postulated that adipocytes might exert a negative effect on hematopoiesis mediated through NP-1. To test this hypothesis, we set up BM adipocytes differentiated into fibroblast-like fat cells (FLFC), which share the major characteristics of primitive unilocular fat cells, as an experimental model. As expected, FLFCs constitutively produced macrophage colony stimulating factor and induced CD34(+) differentiation into macrophages independently of cell-to-cell contact. By contrast, granulopoiesis was hampered by cell-to-cell contact but could be restored in transwell culture conditions, together with granulocyte colony stimulating factor production. Both functions were also recovered when FLFCs cultured in contact with CD34(+) cells were treated with an antibody neutralizing NP-1, which proved its critical implication in contact inhibition. An inflammatory cytokine such as interleukin-1 beta or dexamethasone modulates FLFC properties to restore granulopoiesis. Our data provide the first evidence that primary adipocytes exert regulatory functions during hematopoiesis that might be implicated in some pathological processes. Disclosure of potential conflicts of interest is found at the end of this article.

ADAM metallopeptidase with thrombospondin type 1 motif 2 inactivation reduces the extent and stability of carbon tetrachloride-induced hepatic fibrosis in mice.

UNLABELLED: ADAMTS2 belongs to the "ADAM metallopeptidase with thrombospondin type 1 motif" (ADAMTS) family. Its primary function is to process collagen type I, II, III, and V precursors into mature molecules by excising the aminopropeptide. This process allows the correct assembly of collagen molecules into fibrils and fibers, which confers to connective tissues their architectural structure and mechanical resistance. To evaluate the impact of ADAMTS2 on the pathological accumulation of extracellular matrix proteins, mainly type I and III collagens, we evaluated carbon tetrachloride-induced liver fibrosis in ADAMTS2-deficient (TS2(-/-)) and wild-type (WT) mice. A single carbon tetrachloride injection caused a similar acute liver injury in deficient and WT mice. A chronic treatment induced collagen deposition in fibrous septa that were made of thinner and irregular fibers in TS2(-/-) mice. The rate of collagen deposition was slower in TS2(-/-) mice, and at an equivalent degree of fibrosis, the resorption of fibrous septa was slightly faster. Most of the genes involved in the development and reversion of the fibrosis were similarly regulated in TS2(-/-) and WT mice. CONCLUSION: These data indicate that the extent of fibrosis is reduced in TS2(-/-) mice in comparison with their WT littermates. Inhibiting the maturation of fibrillar collagens may be a beneficial therapeutic approach to interfering with the development of fibrotic lesions.

Histone deacetylases inhibitors as anti-angiogenic agents altering vascular endothelial growth factor signaling.

Angiogenesis is a complex biological process involving the coordinated modulation of many genes. Histone deacetylases (HDAC) are a growing family of enzymes that mediate the availability of chromatin to the transcriptional machinery. Trichostatin-A (TSA) and suberoylanilide hydroxamic acid (SAHA), two HDAC inhibitors known to relieve gene silencing, were evaluated as potential antiangiogenic agents. TSA and SAHA were shown to prevent vascular endothelial growth factor (VEGF)-stimulated human umbilical cord endothelial cells (HUVEC) from invading a type I collagen gel and forming capillary-like structures. SAHA and TSA inhibited the VEGF-induced formation of a CD31-positive capillary-like network in embryoid bodies and inhibited the VEGF-induced angiogenesis in the CAM assay. TSA also prevented, in a dose-response relationship, the sprouting of capillaries from rat aortic rings. TSA inhibited in a dose-dependent and reversible fashion the VEGF-induced expression of VEGF receptors, VEGFR1, VEGFR2, and neuropilin-1. TSA and SAHA upregulated the expression by HUVEC of semaphorin III, a recently described VEGF competitor, at both mRNA and protein levels. This effect was specific to endothelial cells and was not observed in human fibroblasts neither in vascular smooth muscle cells. These observations provide a conspicuous demonstration that HDAC inhibitors are potent anti-angiogenic factors altering VEGF signaling.

TIMP-2 and PAI-1 mRNA levels are lower in aneurysmal as compared to athero-occlusive abdominal aortas.

OBJECTIVE: Significant alterations of the vascular wall occurs in abdominal aortic aneurysm (AAA) and atherosclerotic occlusive disease (AOD) that ultimately may lead to either vascular rupture or obstruction. These modifications have been ascribed to one or a group of proteases, their inhibitors or to the matrix macromolecules involved in the repair process without considering the extent of the observed variations. METHODS: The mRNA steady-state level of a large spectrum of proteolytic enzymes (matrix metalloproteinases: MMP-1, -2, -3, -8, -9, -11, -12, -13, -14; urokinase plasminogen activator: u-PA), their physiological inhibitors (tissue inhibitors of MMPs: TIMP-1, -2, -3; plasminogen activator inhibitor: PAI-1) and that of structural matrix proteins (collagens type I and III, decorin, elastin, fibrillins 1 and 2) was determined by RT-PCR made quantitative by using a synthetic RNA as internal standard in each reaction mixture. The profile of expression was evaluated in AAA (n=7) and AOD (n=5) and compared to non-diseased abdominal (CAA, n=7) and thoracic aorta (CTA, n=5). RESULTS: The MMPs -8, -9, -12 and -13 mostly associated with inflammatory cells were not or barely detected in CAA and CTA while they were largely and similarly expressed in AAA and AOD. Expression of protease inhibitors or structural proteins were only slightly increased in both pathological conditions with the exception of elastin which was reduced. The main significant difference between AAA and AOD was a lower expression of TIMP-2 and PAI-1 in the aneurysmal lesions. CONCLUSIONS: The remodeling of the aortic wall in AAA and AOD involves gene activation of a large and similar spectrum of proteolytic enzymes while the expression of two physiological inhibitors, TIMP-2 and PAI-1, is significantly lower in AAA compared to AOD. The repair process in the aneurysmal disease seems similar to that of the occlusive disease.

Skin physiology in microgravity: a 3-month stay aboard ISS induces dermal atrophy and affects cutaneous muscle and hair follicles cycling in mice.

AIMS: The Mice Drawer System (MDS) Tissue Sharing program was the longest rodent space mission ever performed. It provided 20 research teams with organs and tissues collected from mice having spent 3 months on the International Space Station (ISS). Our participation to this experiment aimed at investigating the impact of such prolonged exposure to extreme space conditions on mouse skin physiology. METHODS: Mice were maintained in the MDS for 91 days aboard ISS (space group (S)). Skin specimens were collected shortly after landing for morphometric, biochemical, and transcriptomic analyses. An exact replicate of the experiment in the MDS was performed on ground (ground group (G)). RESULTS: A significant reduction of dermal thickness (-15%, P=0.05) was observed in S mice accompanied by an increased newly synthetized procollagen (+42%, P=0.03), likely reflecting an increased collagen turnover. Transcriptomic data suggested that the dermal atrophy might be related to an early degradation of defective newly formed procollagen molecules. Interestingly, numerous hair follicles in growing anagen phase were observed in the three S mice, validated by a high expression of specific hair follicles genes, while only one mouse in the G controls showed growing hairs. By microarray analysis of whole thickness skin, we observed a significant modulation of 434 genes in S versus G mice. A large proportion of the upregulated transcripts encoded proteins related to striated muscle homeostasis. CONCLUSIONS: These data suggest that a prolonged exposure to space conditions may induce skin atrophy, deregulate hair follicle cycle, and markedly affect the transcriptomic repertoire of the cutaneous striated muscle panniculus carnosus.

Novel types of mutation responsible for the dermatosparactic type of Ehlers-Danlos syndrome (Type VIIC) and common polymorphisms in the ADAMTS2 gene.

Ehlers-Danlos syndrome (EDS) type VIIC, or dermatosparactic type, is a recessively inherited connective tissue disorder characterized, among other symptoms, by an extreme skin fragility resulting from mutations inactivating ADAMTS-2, an enzyme excising the aminopropeptide of procollagens type I, II, and III. All previously described mutations create premature stop codons leading to a marked reduction in the level of mRNA. In this study, we analyzed the ADAMTS2 cDNA sequences from five patients displaying clinical and/or biochemical features consistent with a diagnosis of either typical or potentially mild form of EDS type VIIC. Three different alterations were detected in the two patients with typical EDS type VIIC. The first patient was homozygous for a genomic deletion causing an in-frame skipping of exons 3-5 in the transcript. In the second patient, the allele inherited from the mother lacks exon 3, generating a premature stop codon, whereas the paternal allele has a genomic deletion resulting in an in-frame skipping of exons 14-16 at the mRNA level. Although the exons 3-5 or 14-16 encode protein domains that have not been previously recognized as crucial for ADAMTS-2 activity, the aminoprocollagen processing was strongly impaired in vitro and in vivo, providing evidence for the requirement of these domains for proper enzyme function. The three other patients with a phenotype with some resemblance to EDS type VIIC only had silent and functionally neutral variations also frequently found in a normal population.

Effects of mild cold shock (25°C) followed by warming up at 37°C on the cellular stress response.

Temperature variations in cells, tissues and organs may occur in a number of circumstances. We report here that reducing temperature of cells in culture to 25°C for 5 days followed by a rewarming to 37°C affects cell biology and induces a cellular stress response. Cell proliferation was almost arrested during mild hypothermia and not restored upon returning to 37°C. The expression of cold shock genes, CIRBP and RBM3, was increased at 25°C and returned to basal level upon rewarming while that of heat shock protein HSP70 was inversely regulated. An activation of pro-apoptotic pathways was evidenced by FACS analysis and increased Bax/Bcl2 and BclX(S/L) ratios. Concomitant increased expression of the autophagosome-associated protein LC3II and AKT phosphorylation suggested a simultaneous activation of autophagy and pro-survival pathways. However, a large proportion of cells were dying 24 hours after rewarming. The occurrence of DNA damage was evidenced by the increased phosphorylation of p53 and H2AX, a hallmark of DNA breaks. The latter process, as well as apoptosis, was strongly reduced by the radical oxygen species (ROS) scavenger, N-acetylcysteine, indicating a causal relationship between ROS, DNA damage and cell death during mild cold shock and rewarming. These data bring new insights into the potential deleterious effects of mild hypothermia and rewarming used in various research and therapeutical fields.

Dissection of Iliac Artery in a Patient With Autosomal Dominant Polycystic Kidney Disease: A Case Report.

Autosomal dominant polycystic kidney disease (ADPKD) is a risk factor for several cardiovascular disorders such as intracranial aneurysm or aortic dissection, preferentially occurring at the thoracic or abdominal level. A 47-year-old man suffering from ADPKD had renal transplantation. Sixteen hours after surgery, he presented with left leg pain. Clinical and ultrasound examination revealed thrombosis of the external left iliac artery. Therefore, we decided to perform intra-arterial angiography to evaluate the possibility of an endovascular treatment. Aorto-femorography showed an obstruction of the external left iliac artery that was found during emergency surgery, consecutive to a dissection, which occurred following the surgery for kidney transplantation. The resected segment of the dissected vessel was analyzed by histology. Collagen fibers organization and density in the adventitia and smooth muscle cells density in the media were similar in the dissected and a normal artery from a healthy donor. By contrast, an almost complete disappearance and fragmentation of elastic lamellae were observed in the media of the dissected artery, most likely responsible for the weakening of the arterial wall and its dissection. Association between ADPKD and single dissection of the iliac artery has been rarely reported. Relationship between inactivation of polycystin/PKD genes and elastic fibers degradation through elevated TGFß signaling and matrix metalloproteinase 2 (MMP2) elastolytic activity, as recently reported in ADPKD, would be worth investigating.

18F-FDG uptake assessed by PET/CT in abdominal aortic aneurysms is associated with cellular and molecular alterations prefacing wall deterioration and rupture.

UNLABELLED: Rupture of abdominal aortic aneurysms (AAAs) leads to a significant morbidity and mortality in aging populations, and its prediction would be most beneficial to public health. Spots positive for uptake of (18)F-FDG detected by PET are found in 12% of AAA patients (PET+), who are most often symptomatic and at high rupture risk. Comparing the (18)F-FDG-positive site with a negative site from the same aneurysm and with samples collected from AAA patients with no (18)F-FDG uptake should allow the discrimination of biologic alterations that would help in identifying markers predictive of rupture. METHODS: Biopsies of the AAA wall were obtained from patients with no (18)F-FDG uptake (PET0, n = 10) and from PET+ patients (n = 8), both at the site positive for uptake and at a distant negative site of the aneurysmal wall. Samples were analyzed by immunohistochemistry, quantitative real-time polymerase chain reaction, and zymography. RESULTS: The sites of the aneurysmal wall with a positive (18)F-FDG uptake were characterized by a strikingly increased number of adventitial inflammatory cells, highly proliferative, and by a drastic reduction of smooth muscle cells (SMCs) in the media as compared with their negative counterpart and with the PET0 wall. The expression of a series of genes involved in the maintenance and remodeling of the wall was significantly modified in the negative sites of PET+, compared with the PET0 wall, suggesting a systemic alteration of the aneurysmal wall. Furthermore, a striking increase of several matrix metalloproteinases (MMPs), notably the MMP1 and MMP13 collagenases, was observed in the positive sites, mainly in the adventitia. Moreover, PET+ patients were characterized by a higher circulating C-reactive protein. CONCLUSION: Positive (18)F-FDG uptake in the aneurysmal wall is associated with an active inflammatory process characterized by a dense infiltrate of proliferating leukocytes in the adventitia and an increased circulating C-reactive protein. Moreover, a loss of SMC in the media and alterations of the expression of genes involved in the remodeling of adventitia and collagen degradation potentially participate in the weakening of the aneurysmal wall preceding rupture.

Rho proteins crosstalk via RhoGDIalpha: At random or hierarchically ordered?

The small GTPases of the Rho family are key signaling molecules regulating a plethora of biological pathways. They can exert diverse, sometimes opposite, contributions to specific cellular processes explaining why their regulation and their crosstalk must be finely tuned. Several mechanisms driving crosstalk between Rho GTPases have been described in the literature. They implicate proteins regulating their activity or common downstream effectors. Among the proteins regulating Rho GTPases cycling, RhoGDIs were viewed until very recently as passive inhibitors. Here, we will focus on recent data supporting a role for RhoGDIalpha in the crosstalk between RhoGTPases and present our results suggesting that "preferential" RhoGDIalpha-mediated crosstalk takes place between closely related Rho GTPases.

Metallothionein-dependent up-regulation of TGF-ß2 participates in the remodelling of the myxomatous mitral valve.

AIMS: Although an excessive extracellular matrix remodelling has been well described in myxomatous mitral valve (MMV), the underlying pathogenic mechanisms remain largely unknown. Our goal was to identify dysregulated genes in human MMV and then to evaluate their functional role in the progression of the disease. METHODS AND RESULTS: Dysregulated genes were investigated by transcriptomic, immunohistochemistry, and western blot analyses of the P2 segment collected from human idiopathic MMV during valvuloplasty (n = 23) and from healthy control valves (n = 17). The most striking results showed a decreased expression of two families of genes: the metallothioneins-1 and -2 (MT1/2) and members of the ADAMTS. The mechanistic consequences of the reduced level of MT1/2 were evaluated by silencing their expression in normal valvular interstitial cells (VICs) cultures. The knock-down of MT1/2 resulted in the up-regulation of transforming growth factor-beta 2 (TGF-ß2). Most importantly, TGF-ß2 was also found significantly increased in MMV tissues. The activation of VICs in vitro by TGF-ß2 induced a down-regulation of ADAMTS-1 and an accumulation of versican as observed in human MMV. CONCLUSION: Our studies demonstrate for the first time that MMV are characterized by reduced levels of MT1/2 accompanied by an up-regulation of TGF-ß2. In turn, increased TGF-ß2 signalling induces down-regulation of aggrecanases and up-regulation of versican, two co-operating processes that potentially participate in the development of the pathology.

RhoGDIα-dependent balance between RhoA and RhoC is a key regulator of cancer cell tumorigenesis.

RhoGTPases are key signaling molecules regulating main cellular functions such as migration, proliferation, survival, and gene expression through interactions with various effectors. Within the RhoA-related subclass, RhoA and RhoC contribute to several steps of tumor growth, and the regulation of their expression affects cancer progression. Our aim is to investigate their respective contributions to the acquisition of an invasive phenotype by using models of reduced or forced expression. The silencing of RhoC, but not of RhoA, increased the expression of genes encoding tumor suppressors, such as nonsteroidal anti-inflammatory drug-activated gene 1 (NAG-1), and decreased migration and the anchorage-independent growth in vitro. In vivo, RhoC small interfering RNA (siRhoC) impaired tumor growth. Of interest, the simultaneous knockdown of RhoC and NAG-1 repressed most of the siRhoC-related effects, demonstrating the central role of NAG-1. In addition of being induced by RhoC silencing, NAG-1 was also largely up-regulated in cells overexpressing RhoA. The silencing of RhoGDP dissociation inhibitor α (RhoGDIα) and the overexpression of a RhoA mutant unable to bind RhoGDIα suggested that the effect of RhoC silencing is indirect and results from the up-regulation of the RhoA level through competition for RhoGDIα. This study demonstrates the dynamic balance inside the RhoGTPase network and illustrates its biological relevance in cancer progression.

Histological and transcriptional study of angiogenesis and lymphangiogenesis in uninvolved skin, acute pinpoint lesions and established psoriasis plaques: an approach of vascular development chronology in psoriasis.

BACKGROUND: Dysregulation of angiogenesis and lymphangiogenesis could participate in psoriasis pathogenesis. Analysis of nascent psoriasis lesions should help at identifying early vascular anomalies. OBJECTIVE: To analyse vascular development, angiogenesis and lymphangiogenesis markers expression in uninvolved skin in psoriatic patients (N), early psoriasis lesions or pinpoints (PP) and psoriasis plaques (PSO). METHODS: Skin biopsies were taken in 17 patients in N and in PSO and/or PP. The mRNA steady-state level of angiogenesis and lymphangiogenesis markers was measured by RT-PCR. Immunohistochemistry was performed for von Willebrand factor, podoplanin, Ki-67 and VEGFR3. Blood (BV) and lymphatic (LV) vessels expansion was measured by computer-assisted morphometry. RESULTS: Clinical and epidermal aspects indicated that PP are intermediate between N and PSO. While total BV area was already increased in PP similarly to PSO as compared to N, LV area in PP was intermediate between N and PSO. Mean LV size was identical in N and PP and increased in PSO, mean BV size in PP being intermediate between N and PSO. VEGF-A 189 variant was increased in PP as compared to N and PSO. As compared to N, angiogenesis markers (VEGF-A isoforms, PlGF, VEGFR2, NRP-1), VEGF-C and NRP-2 were similarly increased in PP and PSO. Keratin 16 and the lymphangiogenesis markers (VEGFR3, prox-1) were intermediate in PP. CONCLUSION: These data suggest that the expansion of lymphatic vessels occurs after blood vascular development in psoriasis. Expansion of BV in PP could be followed by vessel enlargement during progression to PSO, in parallel with a decreased VEGF-A 189/VEGF-A 121 balance in plaques.

RhoA-GDP regulates RhoB protein stability. Potential involvement of RhoGDIalpha.

RhoA plays a significant role in actin stress fibers formation. However, silencing RhoA alone or RhoA and RhoC did not completely suppress the stress fibers suggesting a residual "Rho-like" activity. RhoB, the third member of the Rho subclass, is a shortlived protein barely detectable in basal conditions. In various cell types, the silencing of RhoA induced a strong up-regulation of both total and active RhoB protein levels that were rescued by re-expressing RhoA and related to an enhanced half-life of the protein. The RhoA-dependent regulation of RhoB does not depend on the activity of RhoA but is mediated by its GDP-bound form. The stabilization of RhoB was not dependent on isoprenoid biosynthesis, Rho kinase, extracellular signal-regulated kinase, p38 mitogen-activated kinase, or phosphatidylinositol 3'-OH kinase pathways but required RhoGDIalpha. The forced expression of RhoGDIalpha increased RhoB half-life, whereas its knock-down antagonized the induction of RhoB following RhoA silencing. Moreover, a RhoA mutant (RhoAR68E) unable to bind RhoGDIalpha was significantly less efficient as compared with wild-type RhoA in reversing RhoB up-regulation upon RhoA silencing. These results suggest that, in basal conditions, RhoGDIalpha is rate-limiting and the suppression of RhoA makes it available to stabilize RhoB. Our results highlight RhoGDIalpha-dependent cross-talks that regulate the stability of RhoGTPases.

Newly identified biologically active and proteolysis-resistant VEGF-A isoform VEGF111 is induced by genotoxic agents.

Ultraviolet B and genotoxic drugs induce the expression of a vascular endothelial growth factor A (VEGF-A) splice variant (VEGF111) encoded by exons 1-4 and 8 in many cultured cells. Although not detected in a series of normal human and mouse tissue, VEGF111 expression is induced in MCF-7 xenografts in nude mice upon treatment by camptothecin. The skipping of exons that contain proteolytic cleavage sites and extracellular matrix-binding domains makes VEGF111 diffusible and resistant to proteolysis. Recombinant VEGF111 activates VEGF receptor 2 (VEGF-R2) and extracellularly regulated kinase 1/2 in human umbilical vascular endothelial cells and porcine aortic endothelial cells expressing VEGF-R2. The mitogenic and chemotactic activity and VEGF111's ability to promote vascular network formation during embyonic stem cell differentiation are similar to those of VEGF121 and 165. Tumors in nude mice formed by HEK293 cells expressing VEGF111 develop a more widespread network of numerous small vessels in the peritumoral tissue than those expressing other isoforms. Its potent angiogenic activity and remarkable resistance to proteolysis makes VEGF111 a potential adverse factor during chemotherapy but a beneficial therapeutic tool for ischemic diseases.

Cdc42 downregulates MMP-1 expression by inhibiting the ERK1/2 pathway.

The small GTPases of the Rho family are key intermediates in cellular signalling triggered by activated cell-adhesion receptors. In this study, we took advantage of RNA interference (RNAi) using small interfering RNAs (siRNAs) to define the roles of the best-characterized members of the RhoGTPase family, RhoA, Rac1 and Cdc42, in the control of MMP-1, MMP-2 and type-I-collagen expression in normal human skin fibroblasts (HSFs). A specific and long-lasting repression, up to 7 days after transfection, of the three GTPases was achieved by transient transfection of specific siRNA. The silencing of Cdc42, but not that of RhoA or Rac1, induced a 15-fold increase in MMP-1 secretion. This upregulation was confirmed at the mRNA level and observed with two different siRNAs targeting Cdc42. Such a regulation was also observed in various human cell lines and was rescued by re-expressing wild-type Cdc42 encoded by a construct bearing silent mutations impeding its recognition by the siRNA. By contrast, MMP-2 and type-I-collagen expression was not affected by the individual silencing of each Rho GTPase. Cytokine protein array, enzyme-linked immunosorbent assays and reverse-transcription PCR measurements revealed that ablation of Cdc42 induced an overexpression of interleukin 8 and MCP-1. Although these cytokines are known to induce the expression of MMP-1, we showed that they were not involved in the Cdc42-mediated upregulation of MMP-1. Silencing of Cdc42 also induced an increased phosphorylation of ERK1/2 and p38 MAP kinase. The use of chemical inhibitors on Cdc42-ablated cells revealed that the upregulation of MMP-1 is dependent on the ERK1/2 pathways, whereas the p38 MAP kinase pathway displayed an inhibitory role. Simultaneous knock-down of two or three Rho GTPases allowed us to demonstrate that the RhoA-ROCK pathway was not involved in this regulation but that the silencing of Rac1 reduced the effect of Cdc42 suppression. These data suggest that, in vivo, when cell/extracellular-matrix interactions via integrins induce cytoskeleton organization, MMP-1 expression is maintained at a low level by Cdc42 via a repression of the Rac1 and ERK1/2 pathways. Therefore, Cdc42 contributes to ECM homeostasis and connective tissue integrity.