Atrovastatin-loaded hydrogel affects the smooth muscle cells of human veins

Les pontages veineux restent actuellement un traitement de choix dans les pathologies vasculaires occlusives. Cependant, plusieurs problèmes sont liés à ce type de revascularisation. Premièrement, l'hyperplasie intimale (HI) qui cause une resténose dans 20 à 50% des pontages, conduisant à un échec de la revascularisation. Ce processus est dû à la prolifération et à la migration des cellules musculaires lisses vasculaires vers l'intima, ainsi qu'à une sécrétion de protéines de la matrice extracellulaire conduisant à un épaississement de l'intima, principalement au niveau des anastomoses. Deuxièmement, bien qu'il existe des substances connues pour inhiber l'HI, leur administration systémique répétée est associée à une augmentation de leurs effets secondaires. Aucun dispositif ne permet actuellement la libération d'une telle substance localement au site d'une anastomose vasculaire. Nous avons donc développé un hydrogel d'acide hyaluronique compatible avec une application locale au niveau des anastomoses vasculaires et pouvant être chargé en atorvastatine (ATV) (inhibiteur de la 3-hydroxy-3-methylglutaryl-CoA réductase), substance connue pour inhiber l'HI, dans le but de diminuer le fléau de la resténose. Nous avons tout d'abord testé l'effet de ce gel chargé en ATV sur la prolifération, la migration et la transmigration de cellules musculaires lisses primaires en culture provenant de veines saphènes humaines. Ensuite, nous avons étudié son effet sur différents gènes impliqués dans l'HI. Ceci a permis de montrer que l'ATV diminue la prolifération, la migration et la transmigration des cellules musculaires lisses humaines de façon similaire qu'elle soit ajoutée directement au milieu de culture ou qu'elle soit libérée par l'hydrogel chargé. De même, l'ATV régule de manière simultanée mais différentielle les gènes, en interférant avec le développement de l'HI. Nos expériences montrent que l'HI peut être diminuée in vitro grâce à cet hydrogel d'acide hyaluronique chargé en ATV. Ceci ouvre la porte au développement de futur dispositif permettant de relâcher des substances antisténotiques de façon continue, sur une durée prolongée, et in vivo.

The ciliary smooth muscle electrotransfer: basic principles and potential for sustained intraocular production of therapeutic proteins.

BACKGROUND: We have developed a nonviral gene therapy method based on the electrotransfer of plasmid in the ciliary muscle. These easily accessible smooth muscle cells could be turned into a biofactory for any therapeutic proteins to be secreted in a sustained manner in the ocular media. METHODS: Electrical conditions, design of electrodes, plasmid formulation, method and number of injections were optimized in vivo in the rat by localizing β-galactosidase expression and quantifying reporter (luciferase) and therapeutic (anti-tumor necrosis factor) proteins secretion in the ocular media. Anatomical measurements were performed via human magnetic resonance imaging to design a human eye-sized prototype that was tested in the rabbit. RESULTS: In the rat, transscleral injection of 30 µg of plasmid diluted in half saline (77 mM NaCl) followed by application of eight square-wave electrical pulses (15 V, 10 ms, 5.3 Hz) using two platinum/iridium electrodes, an internal wire and an external sheet, delivered plasmid efficiently to the ciliary muscle fibers. Gene transfer resulted in a long-lasting (at least 5 months) and plasmid dose-/injection number- dependent secretion of different molecular weight proteins mainly in the vitreous, without any systemic exposure. Because ciliary muscle anatomical measurements remained constant among ages in adult humans, an integrated device comprising needle-electrodes was designed and manufactured. Its usefulness was validated in the rabbit. CONCLUSIONS: Plasmid electrotransfer to the ciliary muscle with a suitable medical device represents a promising local and sustained protein delivery system for treating posterior segment diseases, avoiding repeated intraocular injections.

The nuclear hormone receptor PPARγ counteracts vascular calcification by inhibiting Wnt5a signalling in vascular smooth muscle cells.

Vascular calcification is a hallmark of advanced atherosclerosis. Here we show that deletion of the nuclear receptor PPARγ in vascular smooth muscle cells of low density lipoprotein receptor (LDLr)-deficient mice fed an atherogenic diet high in cholesterol, accelerates vascular calcification with chondrogenic metaplasia within the lesions. Vascular calcification in the absence of PPARγ requires expression of the transmembrane receptor LDLr-related protein-1 in vascular smooth muscle cells. LDLr-related protein-1 promotes a previously unknown Wnt5a-dependent prochondrogenic pathway. We show that PPARγ protects against vascular calcification by inducing the expression of secreted frizzled-related protein-2, which functions as a Wnt5a antagonist. Targeting this signalling pathway may have clinical implications in the context of common complications of atherosclerosis, including coronary artery calcification and valvular sclerosis.

Atorvastatin-loaded hydrogel affects the smooth muscle cells of human veins.

Intimal hyperplasia (IH) is the major cause of stenosis of vein grafts. Drugs such as statins prevent stenosis, but their systemic administration has limited effects. We developed a hyaluronic acid hydrogel matrix, which ensures a controlled release of atorvastatin (ATV) at the site of injury. The release kinetics demonstrated that 100% of ATV was released over 10 hours, independent of the loading concentration of the hydrogel. We investigated the effects of such a delivery on primary vascular smooth muscle cells isolated from human veins. ATV decreased the proliferation, migration, and passage of human smooth muscle cells (HSMCs) across a matrix barrier in a similar dose-dependent (5-10 µM) and time-dependent manner (24-72 hours), whether the drug was directly added to the culture medium or released from the hydrogel. Expression analysis of genes known to be involved in the development of IH demonstrated that the transcripts of both the gap junction protein connexin43 (Cx43) and plasminogen activator inhibitor-1 (PAI-1) were decreased after a 24-48-hour exposure to the hydrogel loaded with ATV, whereas the transcripts of the heme oxygenase (HO-1) and the inhibitor of tissue plasminogen activator were increased. At the protein level, Cx43, PAI-1, and metalloproteinase-9 expression were decreased, whereas HO-1 was upregulated in the presence of ATV. The data demonstrate that ATV released from a hydrogel has effects on HSMCs similar to the drug being freely dissolved in the environment.

Endothelial, but not smooth muscle, peroxisome proliferator-activated receptor β/δ regulates vascular permeability and anaphylaxis.

BACKGROUND: Remodeling of quiescent vessels with increases in permeability, vasodilatation, and edema are hallmarks of inflammatory disorders. Factors involved in this type of remodeling represent potential therapeutic targets. OBJECTIVES: We investigated whether the nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR) β/δ, a regulator of metabolism, fibrosis, and skin homeostasis, is involved in regulation of this type of remodeling. METHODS: Wild-type and various Pparb/d mutant mice were used to monitor dermal acute vascular hyperpermeability (AVH) and passive systemic anaphylaxis-induced hypothermia and edema. PPARβ/δ-dependent kinase activation and remodeling of endothelial cell-cell junctions were addressed by using human endothelial cells. RESULTS: AVH and dilatation of dermal microvessels stimulated by vascular endothelial growth factor A, histamine, and thrombin are severely compromised in PPARβ/δ-deficient mice. Selective deletion of the Pparb/d-encoding gene in endothelial cells in vivo similarly limits dermal AVH and vasodilatation, providing evidence that endothelial PPARβ/δ is the major player in regulating acute dermal microvessel remodeling. Furthermore, endothelial PPARβ/δ regulatory functions are not restricted to the skin vasculature because its deletion in the endothelium, but not in smooth muscle cells, also leads to reduced systemic anaphylaxis, the most severe form of allergic reaction, in which an acute vascular response plays a key role. PPARβ/δ-dependent AVH activation likely involves the activation of mitogen-activated protein kinase and Akt pathways and leads to downstream destabilization of endothelial cell-cell junctions. CONCLUSION: These results unveil not only a novel function of PPARβ/δ as a direct regulator of acute vessel permeability and dilatation but also provide evidence that antagonizing PPARβ/δ represents an important strategy to consider for moderating diseases with altered endothelial integrity, such as acute inflammatory and allergic disorders.

The nitric oxide pathway in pig isolated calyceal smooth muscle.

In pig and humans, whose kidneys have a multi-calyceal collecting system, the initiation of ureteral peristalsis takes place in the renal calyces. In the pig and human ureter, recent evidence suggests that nitric oxide (NO) is an inhibitory mediator that may be involved in the regulation of peristalsis. This study was designed to assess whether the NO synthase/NO/cyclic GMP pathway modulates the motility of pig isolated calyceal smooth muscle. Immunohistochemistry revealed a moderate overall innervation of the smooth muscle layer, and no neuronal or inducible NO synthase (NOS) immunoreactivities. Endothelial NOS immunoreactivities were observed in the urothelium and vascular endothelium, and numerous cyclic GMP-immunoreactive (-IR) calyceal smooth muscle cells were found. As measured by monitoring the conversion of L-arginine to L-citrulline, Ca(2+)-dependent NOS activity was moderate. Assessment of functional effects was performed in tissue baths and showed that NO and SIN-1 decreased spontaneous and induced contractions of isolated preparations in a concentration-dependent manner. In strips exposed to NO, there was a 10-fold increase of the cyclic GMP levels compared with control preparations (P < 0.01). It is concluded that a non-neuronal NOS/NO/cyclic GMP pathway is present in pig calyces, where it may influence motility. The demonstration of cyclic GMP-IR smooth muscle cells suggests that NO acts directly on these cells. This NOS/NO/cyclic GMP pathway may be a target for drugs inhibiting peristalsis of mammalian upper urinary tract. Neurourol. Urodynam. 18:673-685, 1999.

Postinfarction heart failure in rats is associated with upregulation of GLUT-1 and downregulation of genes of fatty acid metabolism.

OBJECTIVES: Increasing evidence suggests that left ventricular remodeling is associated with a shift from fatty acid to glucose metabolism for energy production. The aim of this study was to determine whether left ventricular remodeling with and without late-onset heart failure after myocardial infarction is associated with regional changes in the expression of regulatory proteins of glucose or fatty acid metabolism. METHODS: Myocardial infarction was induced in rats by ligation of the left anterior descending coronary artery (LAD). In infarcted and sham-operated hearts the peri-infarction region (5-mm zone surrounding the region at risk), the interventricular septum and the right ventricular free wall were separated for analysis. RESULTS: At 8 and 20 weeks after LAD ligation, the peri-infarction region and the septum exhibited marked re-expression of atrial natriuretic factor [+252+/-37 and +1093+/-279%, respectively, in the septum (P<0.05)] and of alpha-smooth muscle actin [+34+/-10 and +43+/-14%, respectively, in the septum (P<0.05)]. At 8 weeks, when left ventricular hypertrophy was present without signs of heart failure, myocardial mRNA expression of glucose transporters (GLUT-1 and GLUT-4) was not altered, whereas mRNA expression of medium-chain acyl-CoA dehydrogenase (MCAD) was significantly reduced in the peri-infarction region (-25+/-7%; P<0.05). In hearts exhibiting heart failure 20 weeks after infarct-induction there was a change in all three ventricular regions of both mRNA and protein content of GLUT-1 [+72+/-28 and +121+/-15%, respectively, in the peri-infarction region (P<0.05)] and MCAD [-29+/-9 and -56+/-4%, respectively, in the peri-infarction region (P<0.05)]. CONCLUSION: In rats with large myocardial infarction, progression from compensated remodeling to overt heart failure is associated with upregulation of GLUT-1 and downregulation of MCAD in both the peri-infarction region and the septum.

Adult-type rhabdomyosarcoma: analysis of 57 cases with clinicopathologic description, identification of 3 morphologic patterns and prognosis.

Adult-type rhabdomyosarcoma (RMS) has been classically defined as a pleomorphic sarcoma with desmin expression occurring in adult patients. To reevaluate this entity, we analyzed a series of 57 cases using immunohistochemistry for desmin, myogenin, alpha smooth muscle actin, h-caldesmon, pankeratin AE1/AE3, epithelial membrane antigen (EMA), S100 protein, CD34, MDM2, and CDK4. In this series, there were 36 men and 21 women aged from 22 to 87 years (median: 59). Tumors were mainly located in the lower limbs (27 cases), trunk wall (15 cases), and upper limbs (10 cases). Most tumors were deeply located (51/54) with a size from 1 to 30 cm (median: 8 cm). Cases were classified in 3 histologic categories: spindle cell RMS (25 cases), pleomorphic RMS (16 cases), and mixed type (16 cases). Forty-one tumors were grade 3 and 16 grade 2. Immunohistochemistry showed that every case was positive for desmin and myogenin. Alpha smooth muscle actin was positive in 21%, pankeratin AE1/AE3 in 20%, and CD34 in 13.2%. Treatment modalities and follow-up were available in 46 cases. Median follow-up was 60.9 months. Eight patients developed a local recurrence and 16 a distant metastasis with a 5-year overall survival rate of 52.6% and a 5-year metastasis-free survival of 62.9%. The only predictive factor for metastasis was histologic grade. In conclusion, adult-type RMS is a rare sarcoma occurring mainly in the extremities and trunk wall with 2 main histologic patterns, spindle cell, and pleomorphic patterns, which represent the end of the spectrum of a single entity.

Comparative assessment of intimal hyperplasia development after 14 days in two different experimental settings: tissue culture versus ex vivo continuous perfusion of human saphenous vein.

BACKGROUND: Intimal hyperplasia (IH) is a vascular remodeling process which often leads to failure of arterial bypass or hemodialysis access. Experimental and clinical work have provided insight in IH development; however, further studies under precise controlled conditions are required to improve therapeutic strategies to inhibit IH development. Ex vivo perfusion of human vessel segments under standardized hemodynamic conditions may provide an adequate experimental approach for this purpose. Therefore, chronically perfused venous segments were studied and compared to traditional static culture procedures with regard to functional and histomorphologic characteristics as well as gene expression. MATERIALS AND METHODS: Static vein culture allowing high tissue viability was performed as previously described. Ex vivo vein support system (EVVSS) was performed using a vein support system consisting of an incubator with a perfusion chamber and a pump. EVVSS allows vessel perfusion under continuous flow while maintaining controlled hemodynamic conditions. Each human saphenous vein was divided in two parts, one cultured in a Pyrex dish and the other part perfused in EVVSS for 14days. Testing of vasomotion, histomorphometry, expression of CD 31, Factor VIII, MIB 1, alpha-actin, and PAI-l were determined before and after 14days of either experimental conditions. RESULTS: Human venous segments cultured under traditional or perfused conditions exhibited similar IH after 14 days as shown by histomorphometry. Smooth-muscle cell (SMC) was preserved after chronic perfusion. Although integrity of both endothelial and smooth-muscle cells appears to be maintained in both culture conditions as confirmed by CD31, factor VIII, and alpha-actin expression, a few smooth-muscle cells in the media stained positive for factor VIII. Cell-proliferation marker MIB-1 was also detected in the two settings and PAI-1 mRNA expression and activity increased significantly after 14 days of culture and perfusion. CONCLUSION: This study demonstrates the feasibility to chronically perfuse human vessels under sterile conditions with preservation of cellular integrity and vascular contractility. To gain insights into the mechanisms leading to IH, it will now be possible to study vascular remodeling not only under static conditions but also in hemodynamic environment mimicking as closely as possible the flow conditions encountered in reconstructive vascular surgery.

Distribution of neuropeptide Y Y1 receptors in rodent peripheral tissues.

Using a sensitive immunohistochemical technique, the localization of neuropeptide Y (NPY) Y1-receptor (Y1R)-like immunoreactivity (LI) was studied in various peripheral tissues of rat. Wild-type (WT) and Y1R-knockout (KO) mice were also analyzed. Y1R-LI was found in small arteries and arterioles in many tissues, with particularly high levels in the thyroid and parathyroid glands. In the thyroid gland, Y1R-LI was seen in blood vessel walls lacking alpha-smooth muscle actin, i.e., perhaps in endothelial cells of capillaries. Larger arteries lacked detectable Y1R-LI. A distinct Y1R-immunoreactive (IR) reticulum was seen in the WT mouse spleen, but not in Y1R-KO mouse or rat. In the gastrointestinal tract, Y1R-positive neurons were observed in the myenteric plexus, and a few enteroendocrine cells were Y1R-IR. Some cells in islets of Langerhans in the pancreas were Y1R-positive, and double immunostaining showed coexistence with somatostatin in D-cells. In the urogenital tract, Y1R-LI was observed in the collecting tubule cells of the renal papillae and in some epithelial cells of the seminal vesicle. Some chromaffin cells of adrenal medulla were positive for Y1R. The problem of the specificity of the Y1R-LI is evaluated using adsorption tests as well as comparisons among rat, WT mouse, and mouse with deleted Y1R. Our findings support many earlier studies based on other methodologies, showing that Y1Rs on smooth muscle cells of blood vessels mediate NPY-induced vasoconstriction in various organs. In addition, Y1Rs in other cells in parenchymal tissues of several organs suggest nonvascular effects of NPY via the Y1R.

Alpha1-adrenoceptor-dependent vascular hypertrophy and remodeling in murine hypoxic pulmonary hypertension.

Excessive proliferation of vascular wall cells underlies the development of elevated vascular resistance in hypoxic pulmonary hypertension (PH), but the responsible mechanisms remain unclear. Growth-promoting effects of catecholamines may contribute. Hypoxemia causes sympathoexcitation, and prolonged stimulation of alpha(1)-adrenoceptors (alpha(1)-ARs) induces hypertrophy and hyperplasia of arterial smooth muscle cells and adventitial fibroblasts. Catecholamine trophic actions in arteries are enhanced when other conditions favoring growth or remodeling are present, e.g., injury or altered shear stress, in isolated pulmonary arteries from rats with hypoxic PH. The present study examined the hypothesis that catecholamines contribute to pulmonary vascular remodeling in vivo in hypoxic PH. Mice genetically deficient in norepinephrine and epinephrine production [dopamine beta-hydroxylase(-/-) (DBH(-/-))] or alpha(1)-ARs were examined for alterations in PH, cardiac hypertrophy, and vascular remodeling after 21 days exposure to normobaric 0.1 inspired oxygen fraction (Fi(O(2))). A decrease in the lumen area and an increase in the wall thickness of arteries were strongly inhibited in knockout mice (order of extent of inhibition: DBH(-/-) = alpha(1D)-AR(-/-) > alpha(1B)-AR(-/-)). Distal muscularization of small arterioles was also reduced (DBH(-/-) > alpha(1D)-AR(-/-) > alpha(1B)-AR(-/-) mice). Despite these reductions, increases in right ventricular pressure and hypertrophy were not attenuated in DBH(-/-) and alpha(1B)-AR(-/-) mice. However, hematocrit increased more in these mice, possibly as a consequence of impaired cardiovascular activation that occurs during reduction of Fi(O(2)). In contrast, in alpha(1D)-AR(-/-) mice, where hematocrit increased the same as in wild-type mice, right ventricular pressure was reduced. These data suggest that catecholamine stimulation of alpha(1B)- and alpha(1D)-ARs contributes significantly to vascular remodeling in hypoxic PH.

Gene deletion of dopamine beta-hydroxylase and alpha1-adrenoceptors demonstrates involvement of catecholamines in vascular remodeling.

In vitro studies have shown that stimulation of alpha1-adrenoceptors (ARs) directly induces proliferation, hypertrophy, and migration of arterial smooth muscle cells and adventitial fibroblasts. In vivo studies confirmed these findings and showed that catecholamine trophic activity becomes excessive after experimental balloon injury and contributes to neointimal growth, adventitial thickening, and lumen loss. However, past studies have been limited by selectivity of pharmacological agents. The aim of this study, in which mice devoid of norepinephrine and epinephrine synthesis [dopamine beta-hydroxylase (DBH-/-)] or deficient in alpha1-AR subtypes expressed in murine carotid (alpha1B-AR-/- and alpha1D-AR-/-) were used, was to test the hypothesis that catecholamines contribute to wall hypertrophy after injury. At 3 wk after injury of wild-type mice, lumen area and carotid circumference increased significantly, and hypertrophy of media and adventitia was in excess of that needed to restore circumferential wall stress to normal. In DBH-/- and alpha1B-AR-/- mice, increases in lumen area, circumference, and hypertrophy of the media and adventitia were reduced by 50-91%, resulting in restoration of wall tension to nearly normal (DBH-/-) or normal (alpha1B-AR-/-). In contrast, in alpha1D-AR-/- mice, increases in lumen area, circumference, and wall hypertrophy were unaffected and wall thickening remained in excess of that required to return tension to normal. When examined 5 days after injury, proliferation and leukocyte infiltration were inhibited in DBH-/- mice. These studies suggest that the trophic effects of catecholamines are mediated primarily by alpha1B-ARs in mouse carotid and contribute to hypertrophic growth after vascular injury.

The role of MMP-9 and its fibronectin-like domain in tumor growth & invasion : certainties, controversies & discrepancies

Tumors are often compared to wounds that do not heal, where the crosstalk between tumor cells and their surrounding stroma is crucial at all stages of development, from the initial primary growth to metastasis. Similar to wound healing, fibroblasts in the tumor stroma differentiate into myofibroblasts, also referred to as "cancer-associated fibroblasts" (CAFs), primarily, but not exclusively, in response to transforming growth factor-ß (TGF-ß). Myofibroblasts in turn enhance tumor progression by remodeling the stroma. Among molecules implicated in stroma remodeling, matrix metalloproteinases (MMPs), and MMP-g in particular, play a prominent role. However, the mechanisms that regulate MMP-g activation and function remain poorly understood. Recent evidence indicates that tumor cell surface association of MMP-g is an important event in its activation, and more generally in tumor growth and invasion. In the present work we address the potential association of MMP-g activity with cell-surface recruitment to human fibroblasts. We show for the first time that recruitment of MMP-g to the MRC-5 fibroblast cell surface occurs through the fibronectin-like (FN) domain, shared only by MMP-g and MMP-2 among all the MMPs. Functional assays suggest that both the pro- and active form of MMP-g trigger a-smooth muscle actin (aSMA) expression in resting fibroblasts that reflects myofibroblast differentiation, possibly through TGF-ß activation. Moreover, the FN domain of MMP-g inhibits both MMP-g-induced TGF-ß activation and aSMA expression by sequestering MMP-g. Xenograft experiments in NOD/SCID mice using HT1080 fibrosarcoma or MDA-MD231 breast adenocarcinoma cells stably expressing the FN domain of MMP-g revealed no changes in primary tumor growth. However, in the context of metastasis, expression of the FN domain by these same tumor cells dramatically increased their metastatic proclivity whereas expression of wt MMP-g either promoted no change or actually reduced the number of metastases. We observed a decrease of an active form of MMP-g in MDA-MB231 cells overexpressing the FN domain suggesting that the FN domain may inhibit MMP-g activity in Tumors are often compared to wounds that do not heal, where the crosstalk between tumor cells and their surrounding stroma is crucial at all stages of development, from the initial primary growth to metastasis. Similar to wound healing, fibroblasts in the tumor stroma differentiate into myofibroblasts, also referred to as "cancer-associated fibroblasts" (CAFs), primarily, but not exclusively, in response to transforming growth factor-ß (TGF-ß). Myofibroblasts in turn enhance tumor progression by remodeling the stroma. Among molecules implicated in stroma remodeling, matrix metalloproteinases (MMPs), and MMP-g in particular, play a prominent role. However, the mechanisms that regulate MMP-g activation and function remain poorly understood. Recent evidence indicates that tumor cell surface association of MMP-g is an important event in its activation, and more generally in tumor growth and invasion. In the present work we address the potential association of MMP-g activity with cell-surface recruitment to human fibroblasts. We show for the first time that recruitment of MMP-g to the MRC-5 fibroblast cell surface occurs through the fibronectin-like (FN) domain, shared only by MMP-g and MMP-2 among all the MMPs. Functional assays suggest that both the pro- and active form of MMP-g trigger a-smooth muscle actin (aSMA) expression in resting fibroblasts that reflects myofibroblast differentiation, possibly through TGF-ß activation. Moreover, the FN domain of MMP-g inhibits both MMP-g-induced TGF-ß activation and aSMA expression by sequestering MMP-g. Xenograft experiments in NOD/SCID mice using HT1080 fibrosarcoma or MDA-MD231 breast adenocarcinoma cells stably expressing the FN domain of MMP-9 revealed no changes in primary tumor growth. However, in the context of metastasis, expression of the FN domain by these same tumor cells dramatically increased their metastatic proclivity whereas expression of wt MMP-g either promoted no change or actually reduced the number of metastases. We observed a decrease of an active form of MMP-9 in MDA-MB231 cells overexpressing the FN domain suggesting that the FN domain may inhibit MMP-9 activity in those cells and therefore prevent MMP-9-induced activation of TGF-b, which results in increased invasion. Curiously, xenografts of SW480 colorectal adenocarcinoma cells stably expressing the FN domain of MMP-9 displayed reduced growth at both the primary (subcutaneous) injection site and the lungs of NOD/SCID mice, in experimental metastasis assays, whilst the same cells overexpressing wt MMP-9 showed enhanced growth and dissemination. Gelatin zymography of conditioned medium revealed that these effects may be due to the FN domain, which displaces MMP-9 from SW480 cell surface. These observations suggest a dual role of MMP-9 and its FN domain in primary tumor growth and metastasis, underscoring the notion that the effect of MMP-9 on tumor cells may depend on the cell type and highlighting possible protective effects of MMPs in tumor progression.

Neonatal steroids induce a down-regulation of tenascin-C and elastin and cause a deceleration of the first phase and an acceleration of the second phase of lung alveolarization.

Pre- and postnatal corticosteroids are often used in perinatal medicine to improve pulmonary function in preterm infants. To mimic this clinical situation, newborn rats were treated systemically with dexamethasone (Dex), 0.1-0.01 mg/kg/day on days P1-P4. We hypothesized that postnatal Dex may have an impact on alveolarization by interfering with extracellular matrix proteins and cellular differentiation. Morphological alterations were observed on 3D images obtained by high-resolution synchrotron radiation X-ray tomographic microscopy. Alveolarization was quantified stereologically by estimating the formation of new septa between days P4 and P60. The parenchymal expression of tenascin-C (TNC), smooth muscle actin (SMA), and elastin was measured by immunofluorescence and gene expression for TNC by qRT-PCR. After Dex treatment, the first phase of alveolarization was significantly delayed between days P6 and P10, whereas the second phase was accelerated. Elastin and SMA expressions were delayed by Dex treatment, whereas TNC expression was delayed and prolonged. A short course of neonatal steroids impairs the first phase of alveolarization, most likely by altering the TNC and elastin expression. Due to an overshooting catch-up during the second phase of alveolarization, the differences disappear when the animals reach adulthood.

Control of thrombin signaling through PI3K is a mechanism underlying plasticity between hair follicle dermal sheath and papilla cells.

In hair follicles, dermal papilla (DP) and dermal sheath (DS) cells exhibit striking levels of plasticity, as each can regenerate both cell types. Here, we show that thrombin induces a phosphoinositide 3-kinase (PI3K)-Akt pathway-dependent acquisition of DS-like properties by DP cells in vitro, involving increased proliferation rate, acquisition of ;myofibroblastic' contractile properties and a decreased capacity to sustain growth and survival of keratinocytes. The thrombin inhibitor protease nexin 1 [PN-1, also known as SERPINE2) regulates all those effects in vitro. Accordingly, the PI3K-Akt pathway is constitutively activated and expression of myofibroblastic marker smooth-muscle actin is enhanced in vivo in hair follicle dermal cells from PN-1(-/-) mice. Furthermore, physiological PN-1 disappearance and upregulation of the thrombin receptor PAR-1 (also known as F2R) during follicular regression in wild-type mice also correlate with such changes in DP cell characteristics. Our results indicate that control of thrombin signaling interferes with hair follicle dermal cells plasticity to regulate their function.