Exploring the Regulators of Keratinization: Role of BMP-2 in Oral Mucosa.

The oral mucosa functions as a physico-chemical and immune barrier to external stimuli, and an adequate width of the keratinized mucosa around the teeth or implants is crucial to maintaining them in a healthy and stable condition. In this study, for the first time, bulk RNA-seq analysis was performed to explore the gene expression of laser microdissected epithelium and lamina propria from mice, aiming to investigate the differences between keratinized and non-keratinized oral mucosa. Based on the differentially expressed genes (DEGs) and Gene Ontology (GO) Enrichment Analysis, bone morphogenetic protein 2 (BMP-2) was identified to be a potential regulator of oral mucosal keratinization. Monoculture and epithelial-mesenchymal cell co-culture models in the air-liquid interface (ALI) indicated that BMP-2 has direct and positive effects on epithelial keratinization and proliferation. We further performed bulk RNA-seq of the ALI monoculture stimulated with BMP-2 in an attempt to identify the downstream factors promoting epithelial keratinization and proliferation. Analysis of the DEGs identified, among others, IGF2, ID1, LTBP1, LOX, SERPINE1, IL24, and MMP1 as key factors. In summary, these results revealed the involvement of a well-known growth factor responsible for bone development, BMP-2, in the mechanism of oral mucosal keratinization and proliferation, and pointed out the possible downstream genes involved in this mechanism.

Pemafibrate Prevents Rupture of Angiotensin II-Induced Abdominal Aortic Aneurysms.

Background: Abdominal aortic aneurysm (AAA) is a life-threatening disease that lacks effective preventive therapies. This study aimed to evaluate the effect of pemafibrate, a selective peroxisome proliferator-activated receptor alpha (PPARα) agonist, on AAA formation and rupture. Methods: Experimental AAA was induced by subcutaneous angiotensin II (AngII) infusion in ApoE - / - mice for 4 weeks. Pemafibrate (0.1 mg/kg/day) was administered orally. Dihydroethidium staining was used to evaluate the reactive oxygen species (ROS). Results: The size of the AngII-induced AAA did not differ between pemafibrate- and vehicle-treated groups. However, a decreased mortality rate due to AAA rupture was observed in pemafibrate-treated mice. Pemafibrate ameliorated AngII-induced ROS and reduced the mRNA expression of interleukin-6 and tumor necrosis factor-α in the aortic wall. Gelatin zymography analysis demonstrated significant inhibition of matrix metalloproteinase-2 activity by pemafibrate. AngII-induced ROS production in human vascular smooth muscle cells was inhibited by pre-treatment with pemafibrate and was accompanied by an increase in catalase activity. Small interfering RNA-mediated knockdown of catalase or PPARα significantly attenuated the anti-oxidative effect of pemafibrate. Conclusion: Pemafibrate prevented AAA rupture in a murine model, concomitant with reduced ROS, inflammation, and extracellular matrix degradation in the aortic wall. The protective effect against AAA rupture was partly mediated by the anti-oxidative effect of catalase induced by pemafibrate in the smooth muscle cells.

Interaction between S4 and the phosphatase domain mediates electrochemical coupling in voltage-sensing phosphatase (VSP).

Voltage-sensing phosphatase (VSP) consists of a voltage sensor domain (VSD) and a cytoplasmic catalytic region (CCR), which is similar to phosphatase and tensin homolog (PTEN). How the VSD regulates the innate enzyme component of VSP remains unclear. Here, we took a combined approach that entailed the use of electrophysiology, fluorometry, and structural modeling to study the electrochemical coupling in Ciona intestinalis VSP. We found that two hydrophobic residues at the lowest part of S4 play an essential role in the later transition of VSD-CCR coupling. Voltage clamp fluorometry and disulfide bond locking indicated that S4 and its neighboring linker move as one helix (S4-linker helix) and approach the hydrophobic spine in the CCR, a structure located near the cell membrane and also conserved in PTEN. We propose that the hydrophobic spine operates as a hub for translating an electrical signal into a chemical one in VSP.

LCZ696 ameliorates doxorubicin-induced cardiomyocyte toxicity in rats.

Doxorubicin (DOX)-based chemotherapy induces cardiotoxicity, which is considered the main bottleneck for its clinical application. In this study, we investigated the potential benefit of LCZ696, an angiotensin receptor-neprilysin inhibitor against DOX-induced cardiotoxicity in rats and H9c2 cells and determined whether the mechanism underlying any such effects involves its antioxidant activity. Male Sprague-Dawley rats were randomly separated into four groups, each consisting of 15 rats (DOX (1.5 mg/kg/day intraperitoneally for 10 days followed by non-treatment for 8 days); DOX + valsartan (31 mg/kg/day by gavage from day 1 to day 18); DOX + LCZ696 (68 mg/kg/day by gavage from day 1 to day 18); and control (saline intraperitoneally for 10 days). DOX-induced elevation of cardiac troponin T levels on day 18 was significantly reduced by LCZ696, but not valsartan. The DOX-induced increase in myocardial reactive oxygen species (ROS) levels determined using dihydroethidium was significantly ameliorated by LCZ696, but not valsartan, and was accompanied by the suppression of DOX-induced increase in p47phox. LCZ696 recovered the DOX-induced decrease in phosphorylation of adenosine monophosphate-activated protein kinase and increased the ratio of Bax and Bcl-2. In H9c2 cardiomyocytes, LCZ696 reduced DOX-induced mitochondrial ROS generation and improved cell viability more than valsartan. Our findings indicated that LCZ696 ameliorated DOX-induced cardiotoxicity in rat hearts in vivo and in vitro, possibly by mediating a decrease in oxidative stress.

Protective Role of Limitrin in Experimental Autoimmune Optic Neuritis.

Purpose: This study investigated the role of limitrin in the pathogenesis of demyelinating optic neuritis using an experimental autoimmune optic neuritis (EAON) model. Methods: EAON was induced in mice via subcutaneous injection with myelin oligodendrocyte glycoprotein peptide. Limitrin protein and mRNA expression were examined in the optic nerve before and after EAON induction. Proinflammatory cytokine expression profiles and degree of glial activation were compared between wild-type (WT) and limitrin knockout mice by real-time PCR and histologic analysis, respectively, after EAON induction. Plasma limitrin levels in patients with optic neuritis and healthy controls were measured by ELISA. Results: Limitrin expression, observed in astrocytes in the optic nerve of WT mice, was lower in EAON-induced than in naïve WT mice. A comparative analysis of WT and limitrin knockout mice revealed that limitrin deficiency induced more severe neuroinflammation and glial hyperactivation in the optic nerve after EAON induction. Limitrin-deficient astrocytes were more chemotactically responsive to neuroinflammatory stimulation than WT astrocytes. Patients with optic neuritis demonstrated higher plasma limitrin levels than healthy controls (P = 0.0001), which was negatively correlated with visual acuity at the nadir of the optic neuritis attack (r = 0.46, P = 0.036). Conclusions: Limitrin deficiency induced severe neuroinflammation and reactive gliosis in the optic nerve after EAON induction. Our results imply that astrocyte-derived limitrin may protect against neuroinflammation by decreasing immune cell infiltration into the optic nerve. The plasma limitrin level may reflect the extent of blood-brain barrier disruption and provide a valuable biomarker reflecting the severity of optic neuritis.

Lack of collagen α6(IV) chain in mice does not cause severe-to-profound hearing loss or cochlear malformation, a distinct phenotype from nonsyndromic hearing loss with COL4A6 missense mutation.

Congenital hearing loss affects 1 in every 1000 births, with genetic mutations contributing to more than 50% of all cases. X-linked nonsyndromic hereditary hearing loss is associated with six loci (DFNX1-6) and five genes. Recently, the missense mutation (c.1771G>A, p.Gly591Ser) in COL4A6, encoding the basement membrane (BM) collagen α6(IV) chain, was shown to be associated with X-linked congenital nonsyndromic hearing loss with cochlear malformation. However, the mechanism by which the COL4A6 mutation impacts hereditary hearing loss has not yet been elucidated. Herein, we investigated Col4a6 knockout (KO) effects on hearing function and cochlear formation in mice. Immunohistochemistry showed that the collagen α6(IV) chain was distributed throughout the mouse cochlea within subepithelial BMs underlying the interdental cells, inner sulcus cells, basilar membrane, outer sulcus cells, root cells, Reissner's membrane, and perivascular BMs in the spiral limbus, spiral ligament, and stria vascularis. However, the click-evoked auditory brainstem response analysis did not show significant changes in the hearing threshold of Col4a6 KO mice compared with wild-type (WT) mice with the same genetic background. In addition, the cochlear structures of Col4a6 KO mice did not exhibit morphological alterations, according to the results of high-resolution micro-computed tomography and histology. Hence, loss of Col4a6 gene expression in mice showed normal click ABR thresholds and normal cochlear formation, which differs from humans with the COL4A6 missense mutation c.1771G>A, p.Gly591Ser. Therefore, the deleterious effects in the auditory system caused by the missense mutation in COL4A6 are likely due to the dominant-negative effects of the α6(IV) chain and/or α5α6α5(IV) heterotrimer with an aberrant structure that would not occur in cases with loss of gene expression.

Deficiency of CD44 prevents thoracic aortic dissection in a murine model.

Thoracic aortic dissection (TAD) is a life-threatening vascular disease. We showed that CD44, a widely distributed cell surface adhesion molecule, has an important role in inflammation. In this study, we examined the role of CD44 in the development of TAD. TAD was induced by the continuous infusion of ß-aminopropionitrile (BAPN), a lysyl oxidase inhibitor, and angiotensin II (AngII) for 7 days in wild type (WT) mice and CD44 deficient (CD44-/-) mice. The incidence of TAD in CD44-/- mice was significantly reduced compared with WT mice (44% and 6%, p < 0.01). Next, to evaluate the initial changes, aortic tissues at 24 hours after BAPN/AngII infusion were examined. Neutrophil accumulation into thoracic aortic adventitia in CD44-/- mice was significantly decreased compared with that in WT mice (5.7 ± 0.3% and 1.6 ± 0.4%, p < 0.01). In addition, BAPN/AngII induced interleukin-6, interleukin-1ß, matrix metalloproteinase-2 and matrix metalloproteinase-9 in WT mice, all of which were significantly reduced in CD44-/- mice (all p < 0.01). In vitro transmigration of neutrophils from CD44-/- mice through an endothelial monolayer was significantly decreased by 18% compared with WT mice (p < 0.01). Our findings indicate that CD44 has a critical role in TAD development in association with neutrophil infiltration into adventitia.

Engineering an enhanced voltage-sensing phosphatase.

Voltage-sensing phosphatases (VSP) consist of a membrane-spanning voltage sensor domain and a cytoplasmic region that has enzymatic activity toward phosphoinositides (PIs). VSP enzyme activity is regulated by membrane potential, and its activation leads to rapid and reversible alteration of cellular PIP levels. These properties enable VSPs to be used as a tool for studying the effects of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) binding to ion channels and transporters. For example, by applying simple changes in the membrane potential, Danio rerio VSP (Dr-VSP) has been used effectively to manipulate PI(4,5)P2 in mammalian cells with few, if any, side effects. In the present study, we report an enhanced version of Dr-VSP as an improved molecular tool for depleting PI(4,5)P2 from cultured mammalian cells. We modified Dr-VSP in two ways. Its voltage-dependent phosphatase activity was enhanced by introducing an aromatic residue at the position of Leu-223 within a membrane-interacting region of the phosphatase domain called the hydrophobic spine. In addition, selective plasma membrane targeting of Dr-VSP was facilitated by fusion with the N-terminal region of Ciona intestinalis VSP. This modified Dr-VSP (CiDr-VSPmChe L223F, or what we call eVSP) induced more drastic voltage-evoked changes in PI(4,5)P2 levels, using the activities of Kir2.1, KCNQ2/3, and TRPC6 channels as functional readouts. eVSP is thus an improved molecular tool for evaluating the PI(4,5)P2 sensitivity of ion channels in living cells.

Type XVIII Collagen Modulates Keratohyalin Granule Formation and Keratinization in Oral Mucosa.

Epithelial keratinization involves complex cellular modifications that provide protection against pathogens and chemical and mechanical injuries. In the oral cavity, keratinized mucosa is also crucial to maintain healthy periodontal or peri-implant tissues. In this study, we investigated the roles of type XVIII collagen, a collagen-glycosaminoglycan featuring an extracellular matrix component present in the basement membrane, in oral mucosal keratinization. Histological analysis of keratinized and non-keratinized oral mucosa showed that type XVIII collagen was highly expressed in keratinized mucosa. Additionally, a 3D culture system using human squamous carcinoma cells (TR146) was used to evaluate and correlate the changes in the expression of type XVIII collagen gene, COL18A1, and epithelial keratinization-related markers, e.g., keratin 1 (KRT1) and 10 (KRT10). The results showed that the increase in COL18A1 expression followed the increase in KRT1 and KRT10 mRNA levels. Additionally, loss-of-function analyses using silencing RNA targeting COL18A1 mRNA and a Col18-knockout (KO) mouse revealed that the absence of type XVIII collagen induces a dramatic decrease in KRT10 expression as well as in the number and size of keratohyalin granules. Together, the results of this study demonstrate the importance of type XVIII collagen in oral mucosal keratinization.

Collagen XVIII Deposition in the Basement Membrane Zone beneath the Newly Forming Epidermis during Wound Healing in Mice.

The basement membrane (BM) is composed of various extracellular molecules and regulates tissue regeneration and maintenance. Here, we demonstrate that collagen XVIII was spatiotemporally expressed in the BM during skin wound healing in a mouse excisional wound-splinting model. Re-epithelialization was detected at days 3 and 6 post-wounding. The ultrastructure of epidermal BM was discontinuous at day 3, whereas on day 6 a continuous BM was observed in the region proximal to the wound edge. Immunohistochemistry demonstrated that collagen XVIII was deposited in the BM zone beneath newly forming epidermis in day 3 and 6 wounds. Laminin-332, known to be the earliest BM component appearing in wounds, was colocalized with collagen XVIII in the epidermal BM zone at days 3 and 6. The deposition of α1(IV) collagen and nidogen-1 in the epidermal BM zone occurred later than that of collagen XVIII. We also observed the short isoform of collagen XVIII in the epidermal BM zone at day 3 post-wounding. Collectively, our results suggested that collagen XVIII plays a role in the formation of the dermal-epidermal junction during re-epithelialization, and that it is the short isoform that is involved in the early phase of re-epithelialization.

Unripe peach (Prunus persica) extract ameliorates damage from UV irradiation and improved collagen XVIII expression in 3D skin model.

INTRODUCTION: Collagen type XVIII regulates cellular activities of adjacent cells at the dermal-epidermal junction (DEJ). To investigate its possible changes during aging, we compared its mRNA levels and protein localization in skin samples from female participants aged 20-70 years old. In addition, we evaluated the beneficial effects of unripe peach extracts in a 3D skin model. METHODS: Sun-exposed or sun-protected female skin samples were compared by DNA array or by immunohistochemistry for basement membrane components. To evaluate protective effects of fresh unripe peach extract, UV-B irradiated human 3D skin models were incubated in the presence or absence of the extract, followed by measurements of mRNA levels by real-time PCR, or by immunohistochemistry. RESULTS: In aged skin samples, COL18A1 mRNA levels were lower and the protein localization exhibited less intensive signal by anti-collagen type XVIII immunostaining. As observed in the skin tissues, collagen type XVIII exists at the DEJ in the 3D skin model. Fresh unripe peach extract significantly improved mRNA levels and partially localizations of collagen type XVIII, suggesting that fresh unripe peach extract ameliorates DEJ damages caused by UV-B irradiation. CONCLUSION: Collagen type XVIII and fresh unripe peach extract can be promising protective cosmetic strategies against skin aging.

The hydrophobic nature of a novel membrane interface regulates the enzyme activity of a voltage-sensing phosphatase.

Voltage-sensing phosphatases (VSP) contain a voltage sensor domain (VSD) similar to that of voltage-gated ion channels but lack a pore-gate domain. A VSD in a VSP regulates the cytoplasmic catalytic region (CCR). However, the mechanisms by which the VSD couples to the CCR remain elusive. Here we report a membrane interface (named 'the hydrophobic spine'), which is essential for the coupling of the VSD and CCR. Our molecular dynamics simulations suggest that the hydrophobic spine of Ciona intestinalis VSP (Ci-VSP) provides a hinge-like motion for the CCR through the loose membrane association of the phosphatase domain. Electrophysiological experiments indicate that the voltage-dependent phosphatase activity of Ci-VSP depends on the hydrophobicity and presence of an aromatic ring in the hydrophobic spine. Analysis of conformational changes in the VSD and CCR suggests that the VSP has two states with distinct enzyme activities and that the second transition depends on the hydrophobic spine.

Type IV collagen α6 chain is a regulator of keratin 10 in keratinization of oral mucosal epithelium.

Keratinized mucosa is of fundamental importance to maintain healthy gingival tissue, and understanding the mechanisms of oral mucosa keratinization is crucial to successfully manage healthy gingiva. Previous studies have shown a strong involvement of the basement membrane in the proliferation and differentiation of epithelial cells. Therefore, first, to identify the keratinized mucosa-specific basement membrane components, immunohistochemical analysis for the six alpha chains of type IV collagen was performed in 8-week-old mice. No difference in the expression pattern of type IV collagen α1(IV) and α2(IV) chains was observed in the keratinized and non-keratinized mucosa. Interestingly, however, type IV collagen α5(IV) and α6(IV) chains specifically were strongly detected in the keratinized mucosa. To analyze the functional roles of the type IV collagen isoform α6(IV) in oral mucosa keratinization, we analyzed Col4a6-knockout mice. Epithelial developmental delay and low levels of KRT10 were observed in new-born Col4a6-knockout mice. Additionally, in vitro experiments with loss-of function analysis using human gingival epithelial cells confirmed the important role of α6(IV) chain in epithelial keratinization. These findings indicate that α112:α556 (IV) network, which is the only network that includes the α6(IV) chain, is one regulator of KRT10 expression in keratinization of oral mucosal epithelium.

A novel cell exclusion zone assay with a barrier made from room temperature vulcanizing silicone rubber.

OBJECTIVE: To examine the usefulness of room temperature vulcanizing (RTV) silicone rubber as a barrier material for cell exclusion zone assays. METHODS: We created barriers using three types of RTV silicone rubber with differing viscosities. We then assessed the adherence of these barriers to culture dishes and their ease of removal from the dishes. We tested the effect of the newly created barriers on the extracellular matrix (ECM) protein fibronectin by attaching and then removing them from fibronectin-coated culture dishes. We also conducted cell exclusion zone assays with MIO-M1 cells using this new barrier in order to measure cell migration. We used real time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining to measure the effect of fibronectin on MIO-M1 cell migration and the effect of migration (with fibronectin coating) on basic fibroblast growth factor (bFGF) expression in MIO-M1 cells. RESULTS: Of the three types of RTV silicon rubber tested, KE-3495-T was the best in terms of adherence to the dish and ease of removal from the dish. When barrier attachment and removal tests were performed, this rubber type did not have an effect on the fibronectin that coated the dish. In the cell exclusion assay, removal of the barrier revealed that a cell-free area with a distinct margin had been created, which allowed us to conduct a quantitative assessment of migration. Fibronectin significantly promoted the migration of MIO-M1 cells (P = 0.02). In addition, both real time RT-PCR and immunohistological staining indicated that bFGF expression in migrating MIO-M1 cells was significantly higher than that in non-migrating cells (P = 0.03). CONCLUSIONS: RTV silicone rubber can be used to create an effective barrier in cell exclusion zone assays and allows simple and low-cost multi-parametric analysis of cell migration.

The Role of Inverted Internal Limiting Membrane Flap in Macular Hole Closure.

Purpose: To investigate the mechanism of macular hole (MH) closure following the inverted internal limiting membrane (ILM) technique. Methods: We performed the inverted ILM flap surgical technique as an experimental MH model in monkeys, and investigated the process of MH closure immunohistochemically. We then investigated the effects of type IV collagen, fibronectin, and laminin, which are constituent proteins of the ILM, on the proliferation and migration of cultivated Müller cells (MIO-M1). We also investigated the expression of neurotrophic factors and basic fibroblast growth factor (bFGF) in human ILM and MIO-M1 cells, and the effect of MIO-M1 migration on the expression of these factors, via immunohistochemical staining and the real-time reverse transcription polymerase chain reaction. Results: Ten days after inverted ILM flap surgery, the MH had closed and proliferating glial fibrillary acidic protein (GFAP)-positive cells surrounded the ILM. Type IV collagen, fibronectin, and laminin all enhanced the proliferation of MIO-M1 cells, and type IV collagen and fibronectin enhanced the migration of MIO-M1 cells. Neurotrophic factors and bFGF were present on the surface of the human ILM, and MIO-M1 cells produced these factors. Neurotrophic factors and bFGF were expressed to a significantly greater extent by migrating MIO-M1 cells than by these cells in their static state. Conclusions: During MH closure, the ILM functioned as a scaffold for the proliferation and migration of Müller cells, and may promote Müller cell activation. Neurotrophic factors and bFGF produced by activated Müller cells and present on the surface of the ILM may contribute to MH closure.

Suppressive effect of AMP-activated protein kinase on the epithelial-mesenchymal transition in retinal pigment epithelial cells.

The epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells plays a central role in the development of proliferative vitreoretinopathy (PVR). The purpose of this study was to investigate the effect of AMP-activated protein kinase (AMPK), a key regulator of energy homeostasis, on the EMT in RPE cells. In this study, EMT-associated formation of cellular aggregates was induced by co-stimulation of cultured ARPE-19 cells with tumor necrosis factor (TNF)-α (10 ng/ml) and transforming growth factor (TGF)-ß2 (5 ng/ml). 5-Aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR), a potent activator of AMPK, significantly suppressed TNF-α and TGF-ß2-induced cellular aggregate formation (p < 0.01). Dipyridamole almost completely reversed the suppressive effect of AICAR, whereas 5'-amino-5'-deoxyadenosine restored aggregate formation by approximately 50%. AICAR suppressed the downregulation of E-cadherin and the upregulation of fibronectin and α-smooth muscle actin by TNF-α and TGF-ß2. The levels of matrix metalloproteinase (MMP)-2, MMP-9, interleukin-6, and vascular endothelial growth factor were significantly decreased by AICAR. Activation of the mitogen-activated protein kinase and mammalian target of rapamycin pathways, but not the Smad pathway, was inhibited by AICAR. These findings indicate that AICAR suppresses the EMT in RPE cells at least partially via activation of AMPK. AMPK is a potential target molecule for the prevention and treatment of PVR, so AICAR may be a promising candidate for PVR therapy.

COL4A6 is dispensable for autosomal recessive Alport syndrome.

Alport syndrome is caused by mutations in the genes encoding α3, α4, or α5 (IV) chains. Unlike X-linked Alport mice, α5 and α6 (IV) chains are detected in the glomerular basement membrane of autosomal recessive Alport mice, however, the significance of this finding remains to be investigated. We therefore generated mice lacking both α3 and α6 (IV) chains and compared their renal function and survival with Col4a3 knockout mice of 129 × 1/Sv background. No significant difference was observed in the renal function or survival of the two groups, or when the mice were backcrossed once to C57BL/6 background. However, the survival of backcrossed double knockout mice was significantly longer than that of the mice of 129 × 1/Sv background, which suggests that other modifier genes were involved in this phenomenon. In further studies we identified two Alport patients who had a homozygous mutation in intron 46 of COL4A4. The α5 and α6 (IV) chains were focally detected in the glomerular basement membrane of these patients. These findings indicate that although α5 and α6 (IV) chains are induced in the glomerular basement membrane in autosomal recessive Alport syndrome, their induction does not seem to play a major compensatory role.

ADAMTS4 and ADAMTS5 knockout mice are protected from versican but not aggrecan or brevican proteolysis during spinal cord injury.

The chondroitin sulfate proteoglycans (CSPGs) aggrecan, versican, and brevican are large aggregating extracellular matrix molecules that inhibit axonal growth of the mature central nervous system (CNS). ADAMTS proteoglycanases, including ADAMTS4 and ADAMTS5, degrade CSPGs, representing potential targets for ameliorating axonal growth-inhibition by CSPG accumulation after CNS injury. We investigated the proteolysis of CSPGs in mice homozygous for Adamts4 or Adamts5 null alleles after spinal cord injury (SCI). ADAMTS-derived 50-60 kDa aggrecan and 50 kDa brevican fragments were observed in Adamts4-/-, Adamts5-/-, and wt mice but not in the sham-operated group. By contrast Adamts4-/- and Adamts5-/- mice were both protected from versican proteolysis with an ADAMTS-generated 70 kDa versican fragment predominately observed in WT mice. ADAMTS1, ADAMTS9, and ADAMTS15 were detected by Western blot in Adamts4-/- mice' spinal cords after SCI. Immunohistochemistry showed astrocyte accumulation at the injury site. These data indicate that aggrecan and brevican proteolysis is compensated in Adamts4-/- or Adamts5-/- mice by ADAMTS proteoglycanase family members but a threshold of versican proteolysis is sensitive to the loss of a single ADAMTS proteoglycanase during SCI. We show robust ADAMTS activity after SCI and exemplify the requirement for collective proteolysis for effective CSPG clearance during SCI.

Early induction of ADAMTS 1, -4, -5 and -9 in IL-stimulated mouse astrocytes.

AIM: Astrocytes and extracellular matrix molecules have important roles in regulating synaptic functions between neurons in the central nervous system. However, under pathological conditions, these constituents are activated to form glial scar that is thought to be harmful for neuronal regeneration. The aim of this study was to evaluate the expression pattern of ADAMTS1, -4, -5 and -9 in IL-1 stimulated astrocyte cultures obtained from postnatal day zero mouse brains. MATERIAL AND METHODS: Real time PCR analyses were performed. RESULTS: An overexpression of ADAMTS1, -4, -5 and -9 at the 3-h time point after IL-1 stimulation was found. IL-1 stimulation induced aggrecaneses and this effect was time dependent. Maximum increase was detected at 3-h (six fold increase). Interestingly the expression of ADAMTS1 and -4 appeared to be at the highest expression level but the ADAMTS5 and ADAMTS9 expression level was much weaker (three times and two times respectively). CONCLUSION: To the best of our knowledge, this is the first report demonstrating induction of ADAMTS in IL-1 induced astrocytes. Aggrecanases may play a role in tissue destruction in the progression of central nervous system (CNS) injury and they are differentially expressed in mouse CNS, suggesting a critical role in the pathogenesis of CNS injury. This can be a very crucial aetiologic factor for some neuropsychiatric disorders.

Effect of vildagliptin, a dipeptidyl peptidase 4 inhibitor, on cardiac hypertrophy induced by chronic beta-adrenergic stimulation in rats.

BACKGROUND: Heart failure with left ventricular (LV) hypertrophy is often associated with insulin resistance and inflammation. Recent studies have shown that dipeptidyl peptidase 4 (DPP4) inhibitors improve glucose metabolism and inflammatory status. We therefore evaluated whether vildagliptin, a DPP4 inhibitor, prevents LV hypertrophy and improves diastolic function in isoproterenol-treated rats. METHODS: Male Wistar rats received vehicle (n = 20), subcutaneous isoproterenol (2.4 mg/kg/day, n = 20) (ISO), subcutaneous isoproterenol (2.4 mg/kg/day + oral vildagliptin (30 mg/kg/day, n = 20) (ISO-VL), or vehicle + oral vildagliptin (30 mg/kg/day, n = 20) (vehicle-VL) for 7 days. RESULTS: Blood pressure was similar among the four groups, whereas LV hypertrophy was significantly decreased in the ISO-VL group compared with the ISO group (heart weight/body weight, vehicle: 3.2 ± 0.40, ISO: 4.43 ± 0.39, ISO-VL: 4.14 ± 0.29, vehicle-VL: 3.16 ± 0.16, p < 0.05). Cardiac catheterization revealed that vildagliptin lowered the elevated LV end-diastolic pressure observed in the ISO group, but other parameters regarding LV diastolic function such as the decreased minimum dp/dt were not ameliorated in the ISO-VL group. Histological analysis showed that vildagliptin attenuated the increased cardiomyocyte hypertrophy and perivascular fibrosis, but it did not affect angiogenesis in cardiac tissue. In the ISO-VL group, quantitative PCR showed attenuation of increased mRNA expression of tumor necrosis factor-α, interleukin-6, insulin-like growth factor-l, and restoration of decreased mRNA expression of glucose transporter type 4. CONCLUSIONS: Vildagliptin may prevent LV hypertrophy caused by continuous exposure to isoproterenol in rats.