Anti-alpha Smooth Muscle Actin Research Articles

Involvement of Plasminogen Activator Inhibitor-1 in the Pathogenesis of Atopic Cataracts

Further to our previous report of a genetic association between interferon-gamma (IFN-γ) receptor 1 gene and atopic cataract, we investigated the roles of plasminogen activator inhibitor-1 (PAI-1), a fibrosis-related, IFN-γ downstream molecule, in the pathogenesis of atopic cataracts. Cultured lens epithelial cells (LECs) were stimulated by IFN-γ and quantified by PAI-1 mRNA/protein expression. PAI-1 and TGF-β mRNA expression was quantified using cDNA samples obtained from the lens epithelium of atopic cataract patients (n = 7) and of senile cataract patients (n = 8). The anterior capsules obtained from atopic cataracts (n = 9) were immunostained with anti-PAI-1 and anti-alpha smooth muscle actin (α-SMA) antibodies. PAI-1 gene expression was knocked down by PAI-1 siRNA, and α-SMA expression was examined under TGF-β1 stimulation. Expression of α-SMA was examined as a pathological hallmark of anterior subcapsular cataracts, commonly observed in atopic cataracts. The IFN-γ stimulation induced PAI-1 mRNA/protein expression in the LECs from 24 to 48 hours after stimulation. The expression of PAI-1 mRNA and TGF-β1 mRNA was significantly higher in the cDNA samples obtained from the atopic cataracts than those obtained from the senile cataracts. PAI-1-positive immunostaining was observed at the fibrotic lesion of the atopic cataracts, and α-SMA-positive myofibroblasts were observed at the vicinity of the PAI-1-positive lesion in all nine samples examined. PAI-1 gene knockdown resulted in reduced α-SMA expression in the LECs. The findings of this study suggest that the IFN-γ, PAI-1, and TGF-β1 are involved in the pathophysiology of atopic cataracts.

Decreased lymphangiogenesis in mice with osteoarthritis under high fat diet

Purpose: We have reported that lymphatic drainage plays a critical role in limiting the severity of joint tissue injury in mouse model of rheumatoid arthritis by removing catalytic factors and inflammatory cells from interstitial fluids to the local draining lymph nodes. However the implication of lymphatic in the pathogenesis of osteoarthritis (OA) is un-known, especially in OA with diabetic because patients with diabetic often have abnormal micro-vasculature. Currently, there is no method to assess lymphatic draining function in mouse knee as an outcome measure to study lymphatic in OA. Here we examined lymphatic vessels in joint sections from a mouse model of OA with diabetes, and developed a near-infrared lymphatic image technique to monitor lymphatic drainage from knee to local draining lymph nodes in real time. Methods: OA was initiated by a meniscall-ligamentous injury (MLI) procedure in the right knee joint. Type 2 diabetic was induced by feeding mice with high fat (HF, 60% kcal) diet. Controls included mice with sham-operation in the left knee and fed by low fat diet (LF, 10% kcal). 5 wks-old C57/BL/6 mice were placed on HF or LF for 8 wks and then received MLI or sham-operation to initiate OA. Mice were sacrificed 16 wks after the MLI. The knee joints were subjected to histology and IHC stain with anti-podoplanin antibody to identify lymphatic vessels and anti-alpha smooth muscle actin (aSM) antibody to identify aSM covered vessels. A group of 12-wks-old mice were received intra-articular injection of the infrared dye, indocyanine green (ICG), the path and signal intensity of ICG in the knee were recorded under an infrared lymphatic imaging system at 1, 2, 4, 6, and 24 hrs. The ICG clearance was calculated. Results: MLI induced OA-like changes in right knee joints, including increased Chamber score, decreased cartilage thickness and osteophyte formation, which was more severe in the HF group. IHC detected that podoplanin+ vessels had thin wall with large lumen, a feature of lymphatic vessels. These podoplanin+ lymphatic vessels were localized in the connective tissue around joint capsules and periosteam. aSM+ vessels appeared to have thick wall and did not overlap with the podoplanin+ lymphatic vessels, suggesting that they are likely small blood vessels. Interestingly, the density of lymphatic and blood vessels were similar among sham-LF, OA-LF, and sham-HF groups, but remarkably decreased in OA-HF group. Decreased podoplanin+ vessels were more obvious than those of aSM+ vessels. Lymphatic imaging revealed that lymph drains from knee tissues to iliac lymph nodes via a single collecting lymphatic vessel. No lymphatic pulse was observed. The ICG signal intensity declined rapidly after injection. The ICG clearance (vs time 0) was 15% at 2 hrs, 60% at 4 hrs, 80% at 6 hrs and 100% at 24 hrs, indicating an efficient lymphatic drainage in WT mice. Conclusions: HF diet accelerates the progression of OA in a type 2 diabetic mouse model, which is associated with decreased lymphatic vessel formation. The near-infrared lymphatic image can be used to monitor lymphatic draining function in mouse knees longitudinally. The involvement of lymphatic in OA pathology is currently under investigation.

Suppression of Choroidal Neovascularization by Vasohibin-1, a Vascular Endothelium–Derived Angiogenic Inhibitor

PURPOSE. To determine the expression of vasohibin-1 during the development of experimentally induced choroidal neovascularization (CNV) and to investigate the effect of vasohibin-1 on the generation of CNV. METHODS. CNV lesions were induced in the eyes of wild-type (WT) and vasohibin-1 knockout (KO) mice by laser photocoagulation. The expression of vasohibin-1, vascular endothelial growth factor (VEGF), VEGF receptor-1 (VEGFR1), VEGFR2, and pigment epithelial-derived factor (PEDF) was determined by semiquantitative reverse transcription-polymerase chain reaction. The expression of vasohibin-1 was also examined by immunohistochemistry with anti-CD68, anti-alpha smooth muscle actin (αSMA), anti-cytokeratin, and anti-CD31. Vasohibin-1 was injected into the vitreous and the activity and size of the CNV were determined by fluorescein angiography and in choroidal flat mounts. RESULTS. Vasohibin-1 was detected not only in CD31-positive endothelial cells but also in CD68-positive macrophages and αSMA-positive retinal pigment epithelial cells. Strong vasohibin-1 expression was observed at day 28, when the CNV lesions had regressed by histologic examination. The vasohibin-1 level was significantly decreased at day 14 and increased at day 28 after laser application. Significantly less VEGFR2 expression was observed on day 4 after vasohibin-1. The expression of PEDF was not significantly changed by vasohibin-1 injection. Vasohibin-1 injection significantly suppressed the CNV, with no adverse side effects. The CNV lesions in the vasohibin-1-KO mice were significantly larger than those in the WT mice. CONCLUSIONS. The endogenous expression of vasohibin-1 is associated with the natural course of the development of CNV. Intravitreal injections of vasohibin-1 may be a method for inhibiting CNV.

909 INDUCTION OF RADIATION-INDUCED ERECTILE DYSFUNCTION OF RAT AFTER PROSTATE-CONFINED MODERN RADIOTHERAPY

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research1 Apr 2011909 INDUCTION OF RADIATION-INDUCED ERECTILE DYSFUNCTION OF RAT AFTER PROSTATE-CONFINED MODERN RADIOTHERAPY Masaki Kimura, Hui Yan, Zahid Rabbani, Takefumi Satoh, Shiro Baba, Fang-Fang Yin, Thomas Polascik, Craig Donatucci, Zeljko Vujaskovic, and Bridget Koontz Masaki KimuraMasaki Kimura Durham, NC More articles by this author , Hui YanHui Yan Durham, NC More articles by this author , Zahid RabbaniZahid Rabbani Durham, NC More articles by this author , Takefumi SatohTakefumi Satoh Sagamihara, Japan More articles by this author , Shiro BabaShiro Baba Sagamihara, Japan More articles by this author , Fang-Fang YinFang-Fang Yin Durham, NC More articles by this author , Thomas PolascikThomas Polascik Durham, NC More articles by this author , Craig DonatucciCraig Donatucci Durham, NC More articles by this author , Zeljko VujaskovicZeljko Vujaskovic Durham, NC More articles by this author , and Bridget KoontzBridget Koontz Durham, NC More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.800AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Prostate radiation therapy (RT) is a common treatment for localized prostate cancer. Erectile dysfunction (ED) is a common side effect with significant impact on patient quality of life. The mechanisms of RT-induced ED are unclear, as clinical studies are limited and previous animal models do not model current radiation techniques. In this study, we develop an animal model of radiation-induced erectile dysfunction using image-guided three-dimensional (3-D) conformal radiotherapy and begin investigation of mechanisms of RT induced ED. METHODS 60 young adult male rats aged 70–80 days old were divided into age matched sham and RT groups. For RT group, a single 20 Gy fraction to the prostate was delivered by 3D dynamic conformal arc using NovalisTX stereotactic radiotherapy. Penile bulb, shaft, and testes were excluded from treatment fields. Bioassay and intracavernosal pressure (ICP) measurements were conducted at 2, 4, and 9 weeks following RT and in sham animals. After ICP measurements, penile tissues were harvested and evaluated by H&E staining and immunohistochemistry analysis including endothelial nitric-oxide synthase (eNOS) and anti-alpha smooth muscle actin (ASMA). Additionally, the plasma testosterone level of both groups was analyzed using a testosterone ELISA assay kit. Results were compared between RT and sham groups using the Mann-Whitney rank sum test. RESULTS Bioassay and ICP measurements demonstrate statistically significant time-dependant functional impairment of erectile function at 4 and 9 weeks after RT. Plasma testosterone levels were not different between the two groups at any time point (P=0.30, 0.48, and 0.74, respectively). Additionally, immunohistochemistry revealed that expression levels of ASMA and eNOS significantly decreased in cavernous tissue at 9 weeks after RT (P =0.035 and 0.005). All animals received RT could tolerate extremely well without demonstrable toxicities. CONCLUSIONS Our lab has demonstrated the ability to deliver modern radiation therapy to rat prostate using image-guided stereotactic techniques, with time-dependent development of ED. This method can allow investigation into mechanisms of RT-induced ED and its treatment and prevention. Reduction of smooth muscle content and eNOS expression in un-irradiated cavernous tissue is associated with RT induced ED. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e364 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Masaki Kimura Durham, NC More articles by this author Hui Yan Durham, NC More articles by this author Zahid Rabbani Durham, NC More articles by this author Takefumi Satoh Sagamihara, Japan More articles by this author Shiro Baba Sagamihara, Japan More articles by this author Fang-Fang Yin Durham, NC More articles by this author Thomas Polascik Durham, NC More articles by this author Craig Donatucci Durham, NC More articles by this author Zeljko Vujaskovic Durham, NC More articles by this author Bridget Koontz Durham, NC More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract 1431: Nerve growth factor-expressing stromal cells in the microenvironment of hepatic colorectal carcinoma metastasis: Clinical occurrence and functional implications

Abstract Nerve growth factor (NGF) is increased during hepatic regeneration and carcinogenesis, but its role during hepatic metastasis is unknown. A tissue-array collection of metastases from 24 patients who had undergone hepatic excision of colorectal adenocarcinoma metastases was used to investigate NGF and neurotrophin receptor expression by cancer and stromal cells. NGF immunostaining of cancer cells only occurred in 2 out of 24 patients with hepatic metastases, while around 80% of patients had metastases with NGF-expressing stromal cells. Conversely, high affinity TrkA neurotrophin receptor immunoreactivity was mainly concentrated in cancer cells, with low expression occurring in tumor stroma. However, NGF immunostaining of tumor stroma and cancer cell immunostaining with anti-ki67 antibodies did not correlate, suggesting that NGF was not associated to metastatic cell proliferation. Anti-alpha-smooth muscle actin (ASMA) antobodies revealed that majority of metastasis-associated NGF-expressing cells had a myofibroblast phenotype. Interestingly, NGF immunoreactivity was unequivocally localized to desmin-expressing hepatic stellate cells (HSC), a prototypic myofibroblast precursor, and perimetastatic hepatocytes, located at the invasion front of metastases. NGF-expressing hepatocytes had phenotypic features suggesting epithelial-to-mesenchymal transition (snail, vimentin and ASMA overexpression with E-cadherin expression decrease). Consistent with clinical findings, a similar immunostaining pattern was reproduced in the experimental hepatic colonization of C26 and 51b colorectal cells. Moreover, NGF increased by two-fold in the hepatic blood from metastasis-bearing mice and also significantly increased by 2-3-fold in the supernatant of primary cultured HSC given tumor cell-conditioned medium (CM), and hepatocytes given tumor-activated HSC-CM, but not tumor cell-CM. Both NGF secretion and epithelial-to-mesenchymal transition in hepatocytes were TGFbeta-dependent. Recombinant NGF dose-dependently increased chemotactic migration, but not proliferation and adhesion of neurotrophin receptor-expressing cancer cells in vitro. HSC migration-stimulating activity of VEGF and tumor-activated hepatocytes were also NGF-mediated as shown with anti-NGF antibodies. Our results demonstrate that both hepatocyte- and HSC-derived myofibroblasts secrete NGF in the hepatic metastasis microenvironment of human and experimental colorectal carcinoma. The mechanism is TGFbeta-dependent. The migration-stimulating effect of NGF on both cancer and stromal cells suggests its contribution to hepatic metastasis development. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1431.

Angioarchitecture of the venous and capillary system in heart defects induced by retinoic acid in mice

Corrosion casting and immunohistochemical staining with anti-alpha smooth muscle actin and anti-CD34 was utilized to demonstrate the capillary plexus and venous system in control and malformed mouse hearts. Outflow tract malformations (e.g., double outlet right ventricle, transposition of the great arteries, and common truncus arteriosus) were induced in progeny of pregnant mice by retinoic acid administration at day 8.5 of pregnancy. Although control hearts exhibited areas in which capillaries tended to be oriented in parallel arrays, the orientation of capillaries in the respective areas of malformed hearts was chaotic and disorganized. The major branch of a conal vein in control hearts runs usually from the left side of the conus to its right side at the root of the pulmonary trunk and opens to the right atrium below the right auricle; thus, it has a curved course. On the other hand, a conal vein in malformed hearts courses from the left side or from the anterior side of the conus and tends to traverse straight upwards along the dextroposed aorta or along the aortopulmonary groove with its proximal part located outside of the heart. Other cardiac veins in outflow tract malformations are positioned in the same locations as in control hearts. We postulate that the changed location of the conal vein and disorganized capillary plexus result from malformed morphogenesis of the outflow tract and/or a disturbed regulation of angiogenic growth factor release from the adjacent environment.

DEVELOPMENT OF C-KIT IMMUNOPOSITIVE INTERSTITIAL CELLS OF CAJAL IN THE HUMAN STOMACH

Interstitial cells of Cajal (ICC) include several types of specialized cells within the musculature of the gastrointestinal tract (GIT). Some types of ICC act as pacemakers in the GIT musculature, whereas others are implicated in the modulation of enteric neurotransmission. Kit immunohistochemistry reliably identifies the location of these cells and provides information on changes in ICC distribution and density. Human stomach specimens were obtained from 7 embryos and 28 foetuses without gastrointestinal disorders. The specimens were 7–27 weeks of gestational age, and both sexes are represented in the sample. The specimens were exposed to anti-c-kit antibodies to investigate ICC differentiation. Enteric plexuses were immunohistochemically examined by using anti-neuron specific enolase and the differentiation of smooth muscle cells (SMC) was studied with anti-α smooth muscle actin and anti-desmin antibodies. By week 7, c-kit-immunopositive precursors formed a layer in the outer stomach wall around myenteric plexus elements. Between 9 and 11 weeks some of these precursors differentiated into ICC. ICC at the myenteric plexus level differentiated first, followed by those within the muscle layer: between SMC, at the circular and longitudinal layers, and within connective tissue septa enveloping muscle bundles. In the fourth month, all subtypes of c-kit-immunoreactivity ICC which are necessary for the generation of slow waves and their transfer to SMC have been developed. These results may help elucidate the origin of ICC and the aetiology and pathogenesis of stomach motility disorders in neonates and young children that are associated with absence or decreased number of these cells.

Persistence of an Intact Endometrial Matrix and Vessels Structure in Women Exposed to VA-2914, a Selective Progesterone Receptor Modulator

VA-2914 is a selective progesterone receptor modulator with potential contraceptive activity that induces amenorrhea, whereas progestins cause endometrial spotting and bleeding. This abnormal bleeding due to progestins is a consequence of focal stromal proteolysis by an increase in naked vessel size and density. Our objective was to quantify the effects of VA-2914 on endometrial vascularization, fibrillar matrix, and vascular endothelial growth factor (VEGF)-A expression in endometrial biopsies from 41 women before and after 12 wk daily treatment with a placebo, or 2.5, 5, or 10 mg VA-2914. Collagen fibrillar network was stained by silver impregnation. Vessel area, density, and structure were quantified with a computer-assisted image analysis system after double immunostaining using an anti-von Willebrand factor (endothelial cells) and an anti-alpha smooth muscle actin (vascular smooth muscle cells) marker antibody. VEGF-A mRNAs were quantified by RT-PCR and localized by immunohistochemistry. The endometrial vessels, collagen network, and mRNA levels of VEGF-A were identical during the luteal phase at baseline and in VA-2914 treated women. VEGF-A distribution was unchanged. VA-2914 does not alter the endometrial matrix and cells, and does not modify the endometrial vessel morphology as compared with baseline biopsies.

Downhole Sound Radiator for Enhancing Oil Recover

Vascular remodeling is an important pathologic process in vascular injury for various vascular disorders such as atherosclerosis, postangioplasty restenosis and transplant arteriopathy. Recently, pathologic change and the role of bone marrow derived cells were wildly studied in atherosclerosis and restenosis. But the manner of lesion formation in neointima and cell recruitment in vascular remodeling lesion in the present of hypercholesterolemia is not yet fully understood.Double-transgenic mice knockout of LDL receptor gene (LDL -/-) and expressing ubiquitously green fluorescent protein (GFP) were obtained by cross-breeding LDL -/- mice with the GFP-expressing transgenic mice. LDL -/- mice (22 - 24 weeks of age) fed high fat diet containing 1.25% (w/w) cholesterol were subjected to 9Gy irradiation and received bone marrow (BM) cells from the double-transgenic mice. Four weeks later, a nonconstrictive cuff was placed around the right femoral artery. After another 2 weeks, both right and left femoral arteries were harvested and subjected to histochemical analysis. Apoptosis was analyzed in situ using TUNEL assay.Two weeks after cuff placement, atherosclerotic lesions developed in the intima consisting of a massive accumulation of foam cells. The tissue stained with anti-alpha smooth muscle actin (SMA) antibody, showed a number of SMA-positive cells in the intimal lesion area. They were also positive for GFP, indicating that BM-derived cells can differentiate to SMCs in the intima in cuff-induced vascular remodeling lesions. Numerous small vessels in the adventitia as well as the endothelial lining of the intima were positive both for CD31 and GFP. The intima and media showed a large number of TUNEL-positive signals after 2 weeks cuff injury, indicating the presence of apoptosis in vascular remodeling.Atherosclerotic lesions in mice can be developed in the intima after 2 weeks of cuff-induced vascular injury under the hypercholesterolemic conditions. Our data also clearly indicate that bone marrow-derived cells differentiated to smooth muscles and endothelial cells in the formation of these lesions in the presence of hypercholesterolemia.

Endometrial vessel maturation in women exposed to levonorgestrel-releasing intrauterine system for a short or prolonged period of time

Levonorgestrel-releasing intrauterine system (LNG-IUS), although inserted to reduce heavy menstruation, causes irregular early transient bleeding. The objective of the study was to document quantitative changes in endometrial vessels of short- (< or =3 months) and long-term (> or =12 months) LNG users. The area, density and maturation of endometrial vessels were quantified in 19 endometrial biopsies of women with LNG-IUS and in 10 normally ovulating patients during mid-luteal phase. Vessel maturation was evaluated by double immunostaining using anti-von Willebrand factor (endothelial cell marker) and anti-alpha Smooth Muscle Actin (vascular smooth muscle cells) antibodies. Vessel area, number and density were quantified with a novel computer-assisted image analysis system. Endometrium exposed to LNG-IUS for 1-3 months displayed a 11.5-fold increase in small naked vessel number. The partially mature vessel (alphaSMA partially positive) number increased six times. After long-term LNG-IUS treatment, the immature and partially mature vessel number remained four times higher than in the control group. Vessel area and density also increased dramatically in a time-dependent pattern with LNG-IUS use. Levonorgestrel affects blood vessel number, area, density and maturation in a time-dependent pattern that may explain the early transient increase in breakthrough bleeding with the LNG-IUS.

Restoration of bladder contraction by bone marrow transplantation in rats with underactive bladder

We attempted to increase bladder contraction by bone marrow cell transplantation in rats with underactive bladder due to bladder outlet obstruction (BOO). Twelve female rats were anesthetized with halothane to create BOO. After 1 month, the urethral obstruction was removed and they were divided into a transplant group and a sham-operated group (n = 6 each). Bone marrow cells (1 x 10(7) / 0.2 mL) isolated from green fluorescent protein transgenic rats were injected into the bladder wall of the transplant group. Rats from the sham-operated group received injection of culture medium alone. One month after transplantation, isovolumetric cystometry parameters and histological features of bladder were observed as well as intact control rats (n = 6). The amplitude of bladder contractions was larger and the interval between contractions was shorter in the transplant group than the sham-operated group, and there were no differences in these parameters between the transplant group and the control group. Some green fluorescent muscle layers were found in the bladder wall of the transplant group, and these layers were also labeled by anti alpha-smooth muscle actin antibody. These results suggest that transplanted bone marrow cells may improve bladder contractility by differentiating into smooth muscle-like cells.

Increased arterial wall volume following chronic reduction of blood flow

Vascular networks respond to chronic alterations in blood supply by structural remodeling. A previous study showed that the diameter of the mouse gracilis artery increased transiently following a permanent reduction in blood flow, with a peak at 21 days. Here, the objective was to investigate whether the diameter increase is accompanied by increased vessel wall mass in the same experimental setting. Blood flow reduction was induced by removing a portion of the saphenous artery, one of the two blood supplies of the gracilis artery. After 21 days, the vasculature was perfused with India ink for diameter measurements, embedded in paraffin, sectioned and processed for immunocytochemistry using fluorescently labeled anti smooth muscle alpha-actin antibody and bisbenzimidazole (BBI) to mark cell nuclei. The remodeled vessel wall volume was almost doubled relative to the control, and the number of smooth muscle cell nuclei increased significantly. The results are consistent with the concept that outward remodeling causes increased circumferential wall stress, which then stimulates vessel wall growth to restore circumferential stress to near its initial level. Supported by AHA Grant 0010189Z and NIH Grants HL34555 and HL63732.

Rat liver fibrosis regresses better with pegylated interferon α2b and ursodeoxycholic acid treatments than spontaneous recovery

Fibrosis and cirrhosis are common complications of chronic liver diseases. An imbalance between fibrogenesis and fibrolysis results in scarring of the liver parenchyma. We aimed to investigate the possible antifibrotic effectiveness of a newly modified interferon molecule peginterferon alpha2b (PEG-IFNalpha2b) which has better antiviral activity, and ursodeoxycholic acid (UDCA). Liver fibrosis was established on 60 male Sprague Dawley rats with CCl4 in 12 weeks. After cessation of CCl4 Group I was left for spontaneous recovery. Group II was treated with PEG-IFN 1.5 microg/kg/week, Group III with UDCA 25 mg/kg/day and Group IV with combination of both drugs. All rats were killed at week 16. Histopathologic fibrosis scores, tissue hydroxyproline, TIMP-1 and MMP-13 levels were determined. Hepatic stellate cell apoptosis was detected by dual staining with TUNEL technique and anti-alpha smooth muscle actin. Fibrosis scores were lower in Group II, III and IV than Group I (p<0.05 for group I vs. II and III; p<0.01 for group I vs. IV). Tissue hydroxyproline levels were significantly decreased in Group II, III and IV when compared to Group I (p<0.05 for group I vs. II, p<0.01 for group I vs. III and IV). Lower liver TIMP-1 and higher MMP-13 levels were measured in Group II, III, and Group IV than Group I (p<0.01 for TIMP-1 and p<0.01, for MMP). Activated HSC apoptosis was significantly increased in Group II, III and IV when compared to Group I (p<0.01, for all). There was significantly higher apoptosis in Group II than Group III and IV (p<0.01). Treatment with both PEG-IFNalpha2b and UDCA improved CCl4 induced rat liver fibrosis. Significantly higher effects were obtained using these agents in combination.

Localization of FGF-6 and FGFR-4 during prenatal and early postnatal development of the mouse sublingual gland

A number of fibroblast growth factors (FGFs) are involved in regulatory mechanisms of the salivary gland development. However, the role of FGF-6 unique in myogenic cells has not been elucidated in the developing sublingual gland. In the present study, temporo-spatial expression of FGF-6 and its receptor (FGFR)-4, in conjunction with some related histo-chemical properties, were investigated in the sublingual gland of the prenatal and early postnatal mice. The earliest expression of both FGF-6 and FGFR-4 was detected in immature acinar cells at gestational day 17 (GD17). The staining intensity increased gradually and some acinar cells showed a distinct staining at postnatal day 0 (PD0). The immunopositive cells had a relatively round profile and were assumed to be acinar cells. The positive staining decreased thereafter and disappeared completely by PD11. To confirm the identity of cells positive for FGF-6, double immunolabeling with anti-alphasmooth muscle actin (alphaSMA) and anti-FGF-6 antibodies was performed. The positive staining of alphaSMA, a marker of myoepithelial cells, was detected in the flattened cells surrounding the acini but not in the cells positive for FGF-6. The staining properties of secretory granules in acinar cells were also examined with periodic acid-Shiff (PAS) and alcian blue (AB). PAS-positive granules abundant in the late gestational stages (GD17 to PD0) began to be replaced with AB-positive mucous granules at early neonatal days (PD0-3), when the FGF-6/FGFR-4 expression was the strongest. These findings suggest that FGF-6/FGFR-4 might be involved in the changes of secretory granule content of acinar cells in the sublingual gland during the late gestational and early neonatal stages.

Healing Process at the Flap Edge in Its Influence in the Development of Corneal Ectasia After LASIK

Purpose: Post–laser in situ keratomileusis (LASIK) corneal ectasia is a progressive deformation of the gross corneal anatomy that occurs after surgery. However, this is a rare event even after deep lamellar keratoplasty. We hypothesize that the strength of the lamellar keratoplasty wound is derived from the sutures that enhance the wound edge healing response. This study compares, in a rabbit model, the stability of previously sutured and unsutured microkeratome flaps. Methods: Unilateral 160-μm-thick LASIK flaps using a mechanical microkeratome was performed in 20 rabbit eyes. Animals were then divided in two groups: In group A, the flap was left without sutures. In group B, the flap was sutured with 12 interrupted 10/0 nylon stitches that were removed after 3 weeks under general anesthesia. Six weeks after surgery, all rabbits had corneal topographies performed at their baseline intraocular pressure (IOP) (14 mmHg) and at two artificially increased pressures (25 and 45 mmHg) using an anterior chamber maintainer implanted in the inferior limbal area. The animals were humanely euthanized, and immunohistological analysis of the corneas was performed. Results: A delta K1 value, which indicates the difference in the simulated keratometric value at baseline and the one measured at 25 mmHg, was calculated for all eyes. It showed a mean steepening effect of 2.74 D ± 0.38 D in group A compared with 1.08 D ± 0.27 D in group B (p < 0.05). Similarly, a delta K2 value, which indicates the difference in the simulated keratometric value at baseline and the one obtained at 45 mmHg, was registered. It showed a mean steepening effect of 3.02 D ± 0.87 D in group A compared with 0.75 D ± 0.44 D in group B (p < 0.05). Six weeks after surgery, the peripheral flap interface in group B consisted of 14.3% ± 4.15% of positive monoclonal mouse anti-alpha smooth muscle actin (α-SMA) cells compared with 4.18 ± 3.76% in group A (p < 0.05). Conclusions: The addition of sutures in the corneal flap after LASIK appears to reduce the amount of corneal steepening when the IOP is artificially increased up to 25 mmHg in this rabbit model. Our results suggest that an increase in the amount of myofibroblastas induced by the sutures may be responsible for this behavior. Corneal ectasia may be related to the clinically observed lack of corneal wound-healing at the edge of the flap that allows the cornea to bulge. By stimulating a stronger wound-healing response at the edge of the flap, the cornea may better resist steepening under increased IOP conditions and improve the long-term stability of LASIK surgery in borderline thin corneas.

Coordinate cell-surface expression of matrix metalloproteinases and their inhibitors on cancer-associated myofibroblasts from malignant ascites in patients with gastric carcinoma

The crucial role of tumor stroma in cancer cell invasion has been described in human carcinoma tissues. However, myofibroblastic invasion remains largely unexplored in malignant ascites. Purpose of this study is to investigate the spatial localization or regulation of matrix metalloproteinases (MMP-2, -7 -9, MT1-MMP) and their inhibitors (TIMP-2 and -4) on myofibroblasts from malignant ascites in 20 patients with gastric carcinoma. The quantitative flow cytometric analysis of MMPs or TIMPs on myofibroblasts was based on the percentage of double positive cells defined by anti MMPs or anti TIMPs, and anti alpha-smooth muscle actin (alpha-SMA) antibodies. The results clearly showed that the coordination of the high level of cell-surface expression of secreted MMPs and TIMPs was noted on the alpha-SMA+ myofibroblasts. The finding suggests the possible formation of ternary complex, MT1-MMP/TIMPs/MMPs on the cells. The events might be a cause and result of activation processing of MMPs on the cells. This study provides the presence of invasive myofibroblasts with activated MMPs in close association with MMPs+ and TIMPs+ cancer cells and tumor-infiltrating lymphocytes from malignant ascites, emphasizing the importance of molecular cross-talk in tumor-host microenvironment for cancer invasion, metastasis and progression.

Epidermal dermal junction and spots in human skin

Epidermal dermal junction and spots in human skin

Embryonic development of the ganglion plexuses and the concentric layer structure of human gut: a topographical study.

In this study, we performed a detailed topographical study on the development of ganglion plexuses and the smooth muscle layers of human embryonic and fetal gut. Neuron and glia differentiation was investigated with anti-PGP9.5 and anti-S100 antibodies respectively. The differentiation of smooth muscle and interstitial cells of Cajal (ICC) was studied with anti-smooth muscle alpha-actin and anti-C-Kit antibodies respectively. By week 7, rostro-caudal neural crest cell (NCC) colonization of the gut was complete, and NCCs have differentiated into neurons and glia. At the foregut, neurons and glia were aggregated into ganglion plexus in the myenteric region, and the longitudinal and circular muscle layers have started to differentiate; however, neurons and glia were not found in the submucosa. At the hindgut, neurons and glia were dispersed within the mesenchyme. Myenteric plexus, longitudinal and circular muscle layers formed along the entire gut by week 9. Scattered and individual neurons and glia, and small ganglion plexuses were detected in the foregut and midgut submucosa by week 12. Ganglion plexus was not seen in the hindgut submucosa until week 14. Muscularis mucosae was formed at the foregut and midgut by week 12 but was only discernible at the hindgut 2 weeks later. As the gut wall developed, ganglion plexus increased in size with more neurons and glia, and the formation of intra-plexus nerve fascicle. ICCs were localized in the ganglion plexus as early as week 7. ICCs were initially dispersed in the plexus and were preferentially localized at the periphery of the plexus by week 20. The specification of the annular layers of human embryonic and fetal gut follows a strict spatio-temporal pattern in a rostro-caudal and centripetal manner suggesting that interaction between (1) homotypic and/or heterotypic cells; and (2) cells and the extracellular matrix is critical for the embryonic development of the gut mesenchyme and the enteric nervous system.

Immature tumor angiogenesis in high-grade and high-stage renal cell carcinoma

Objectives To investigate the correlation between pathologic findings and maturation of the tumor neovasculature of renal cell carcinoma by immunohistochemical studies. Methods Formalin-fixed and paraffin-embedded specimens from 25 randomly selected patients with renal cell carcinoma were stained with mouse monoclonal antibodies, anti-human CD31, anti-alpha smooth muscle actin (αSMA), and anti-human calponin by the indirect immunoperoxidase method. The microvessels were counted in six areas with the higher number of microvessels in each patient at 200× magnification (0.255 mm 2 per area). Results The number of CD31-positive microvessels in grade 3 tumors was significantly lower than those in grade 1 or 2 tumors ( P = 0.003222 and P = 0.043217, respectively). The CD31-positive microvessel counts of those of higher stage, tumor size greater than 4.5 cm, or non-clear cell type were significantly lower than tumors of lower stage, size less than 4.6 cm, or clear cell type. In the grade 3 tumors, the expression ratio of the number of αSMA-positive microvessels to the number of CD31-positive microvessels was significantly decreased compared with grade 1 or 2 tumors ( P = 0.000011 and P = 0.000000, respectively). The expression of calponin in the tumor neovasculature was not observed. The expression ratios of the number of αSMA-positive microvessels to the number of CD31-positive microvessels in higher stages, larger tumor sizes, or non-clear cell types were significantly decreased. Conclusions The tumor neovasculature of high-grade and high-stage tumors was immature. These results imply that high-grade tumors of renal cell carcinomas may be susceptible to antiangiogenesis therapy inducing apoptosis of immature tumor vessels.

Expression of the endothelial receptor tyrosine kinase Tie2 in lobular capillary hemangioma of the oral mucosa: an immunohistochemical study.

Lobular capillary hemangioma (LCH) usually occurs in the skin or mucous membranes as a rapidly growing red nodule. LCH is one of the most common vascular lesions in the oral mucosa. Tie2 is a novel, human endothelial receptor tyrosine kinase which may play an important role in blood vessel formation. In this study, we investigated its immunohistochemical expression in LCH of the oral mucosa. Double immunostaining with anti-CD34 and antialpha smooth muscle actin (SMA) antibodies was performed to characterize the cellular expression of Tie2 in 15 cases of this condition. We found Tie2 immunoreactivity in the ovoid cells only, which were frequently intermingled with alphaSMA-positive cells, especially in the hypercellular portion of LCH. These results suggest that the expression of Tie2 in ovoid cells and the interaction between ovoid cells and alphaSMA-positive cells play an important part in the development and progression of LCH.