Alpha-smooth Muscle Actin Staining Research Articles

KIF18A inactivates hepatic stellate cells and alleviates liver fibrosis through the TTC3/Akt/mTOR pathway.

Activation of hepatic stellate cells (HSCs) has been demonstrated to play a pivotal role in the process of liver fibrogenesis. In this study, we observed a decrease in the expression of KIF18A in fibrotic liver tissues compared to healthy liver tissues, which exhibited a negative correlation with the activation of HSCs. To elucidate the molecular mechanisms underlying the involvement of KIF18A, we performed in vitro proliferation experiments and established a CCl4-induced liver fibrosis model. Our results revealed that KIF18A knockdown enhanced HSCs proliferation and reduced HSCs apoptosis in vitro. Mouse liver fibrosis grade was evaluated with Masson's trichrome and alpha-smooth muscle actin (α-SMA) staining. In addition, the expression of fibrosis markers Col1A1, Stat1, and Timp1 were detected. Animal experiments demonstrated that knockdown of KIF18A could promote liver fibrosis, whereas overexpression of KIF18A alleviated liver fibrosis in a CCl4-induced mouse model. Mechanistically, we found that KIF18A suppressed the AKT/mTOR pathway and exhibited direct binding to TTC3. Moreover, TTC3 was found to interact with p-AKT and could promote its ubiquitination and degradation. Our findings provide compelling evidence that KIF18A enhances the protein binding between TTC3 and p-AKT, promoting TTC3-mediated ubiquitination and degradation of p-AKT. These results refine the current understanding of the mechanisms underlying the pathogenesis of liver fibrosis and may offer new targets for treating this patient population.

Time-restricted feeding has a limited effect on hepatic lipid accumulation, inflammation and fibrosis in a choline-deficient high-fat diet-induced murine NASH model.

Nonalcoholic steatohepatitis (NASH) occurs worldwide and is characterized by lipid accumulation in hepatocytes, hepatic inflammation, fibrosis, and an increased risk of cirrhosis. Although a major proportion of NASH patients exhibit obesity and insulin resistance, 20% lack a high body mass and are categorized as "non-obese NASH". Time-restricted feeding (TRF), limiting daily food intake within certain hours, improves obesity, lipid metabolism, and liver inflammation. Here, we determined whether TRF affects NASH pathology induced by a choline-deficient high-fat diet (CDAHFD), which does not involve obesity. TRF ameliorated the increase in epididymal white adipose tissue and plasma alanine transaminase and aspartate transaminase levels after 8 weeks of a CDAHFD. Although gene expression of TNF alpha in the liver was suppressed by TRF, it did not exhibit a suppressive effect on hepatic lipid accumulation, gene expression of cytokines and macrophage markers (Mcp1, IL1b, F4/80), or fibrosis, as evaluated by Sirius red staining and western blot analysis of alpha-smooth muscle actin. A CDAHFD-induced increase in gene expression related to fibrogenesis (Collagen 1a1 and TGFβ) was neither suppressed by TRF nor that of alpha-smooth muscle actin but was increased by TRF. Our results indicated that TRF has a limited suppressive effect on CDAHFD-induced NASH pathology.

Abstract P1140: Acute Mechanosensitive Adaptation To Phenylephrine In Cardiomyocytes And Fibroblasts

Mechanosensitive pathways are fine tuned to the physiological environment of the cell. Adrenergic stimulation induces cell type specific responses in the heart, including cardiomyocyte hypertrophy and fibroblast proliferation. We hypothesized acute induction of hypertrophy by phenylephrine (PHE) would activate mechanosensitive pathways in fibroblasts and myocytes, revealing novel molecular features of early hypertrophic adaptation. Male mice were subcutaneously injected with 20 mg/kg of PHE or PBS every other day for one week. Echocardiography revealed significant cardiac hypertrophy with increased ejection fraction (72.45% + 6.15 PHE v 56.32 + 5.32 PBS, p<0.0001) and LV wall thickness (2.25 mm + 0.29 PHE v 1.62 + 0.13 PBS, p<0.0001), along with diastolic impairment, as measured by elevated E/e’ ratio (37.25 + 7.29 PHE v 23.67 + 3.62 PBS, p<0.0001). Interestingly, histological analysis showed increased fibrotic deposition and cardiomyocyte cross-sectional area was preferential increased in fibrotic areas (265.5 um 2 + 67.5 PHE fibrotic v 195.1 + 47.46 PHE non-fibrotic v 165.6 + 46.96 PBS). While cardiac fibroblasts from PHE-treated animals did not show alpha smooth muscle actin staining, a traditional marker for activated myofibroblasts, fibroblasts from PHE-treated mice showed increased f-actin stress fibers and nuclear localization of Mrtfa, a mechanosensitive transcriptional coactivator. We performed RNA-seq on isolated cardiomyocytes, demonstrating activation of myocyte-specific responses to increased pressure ( Des, Nppa ), as well as activation of fibrotic genes ( Tgfbr1, Tgfbr2, Tgfb2 ) in myocytes . We sought to investigate mechanosensitive pathways initiated by PHE treatment in cardiomyocytes and identified transcriptional changes in Ankrd1, a Titin binding protein which translocates to the nucleus upon stress. We demonstrate a PHE induced increase in Ankrd1 protein expression and nuclear localization (nuclear area occupancy, as measured by microscopy, 17.04% + 8.76 PHE v 11.91% + 9.134 PBS, p=0.002) in cardiomyocytes. Together these findings demonstrate distinct acute phenotyptic adaptation to adrenergic stress in myocytes and fibroblasts, revealing cell type-specific mechanosensitive signaling mechanisms.

Sympathetic Spleen Denervation Attenuate Pulmonary Hypertension by Decreasing Lung Infiltration of Hematopoietic Stem Cells And Neuroinflammation

Background: Pulmonary Hypertension (PH) is a severe disease characterized by increased pulmonary arterial pressure that ultimately leads to right heart failure. Previous studies have indicated PH as a disease that affects several other organ systems in addition to the lungs. Of particular interest has been the brain and its contribution to the pathogenesis of PH. Neural control of immunity and inflammation is well-established in the literature, whereby sympathetic-mediated emergency granulopoiesis from the spleen is a critical source for the inflammatory process in different diseases. Aim: Investigate the hypothesis of a feedforward loop involving increased sympathetic activity and hematopoietic stem cells (HSCs). In this hypothesis, infiltration of HSC to the brain contributes to neuroinflammation, maladaptive neuroplasticity in preautonomic neurons, and consequently, autonomic disbalance, which contributes to increased inflammatory response and severity of PH. Method: Sympathetic denervation of spleen was performed in C57BL6-J mice (n=40) using absolute ethanol/saline repeatedly applied in the splenic hilum. Following completion of either splenic denervation or sham surgery, animals were allowed to recover for 6 weeks before exposure to either Chronic Hypoxia (CH-PH; FIO2 10%) or normoxia (room air, FIO2 21%) for 4 weeks. Then, right ventricular systolic pressure (RVSP) was recorded, followed by harvest of tissues. Alpha-smooth muscle actin (αSMC) and Masson trichrome staining in the lungs was used for characterization of pulmonary hypertension. Immune cell profile in the lung, spleen and brain was performed by flow cytometry. Iba1 staining was used to label microglia/macrophage cells. Together, histological and flow data were used to evaluate whether the neuroinflammation observed in CH-PH is dependent/independent of sympathetic-mediated infiltration of myeloid precursors from the circulation to preautonomic nuclei such as the paraventricular nucleus of the hypothalamus (PVN). TH staining was performed in the spleen of all animals to confirm the spleen denervation. Result: Sympathetic spleen denervation attenuated the increase of RVSP, Iba1+ cells within the PVN and proliferation of αSMC and collagen in the lungs induced by CH. Furthermore, sympathetic denervation also blunted the changes in the catecholamine metabolism in PMN-MDSC cells within the lung and altered TREM2+ microglia cells’ response to CH. Conclusion: Our previous experiments demonstrated that the exposure of chimeric mice with GFP bone marrow cells to chronic hypoxia illustrates the translocation of these cells into the brain of PH animals. Current experiments show that sympathetic spleen denervation attenuates the infiltration of hematopoietic cells to the PVN and PH development. Together, these results indicate that hematopoietic cells may contribute directly to lung inflammation as well as to the dysfunctional neuro-immune axis observed in PH. NIH grant HL102033, HL110170, HL142776; AHA 20POST35210516 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract C048: Benzodiazepines modify the pancreatic ductal adenocarcinoma tumor microenvironment

Abstract Benzodiazepines (BZDs) are a class of drugs commonly prescribed to pancreatic ductal adenocarcinoma (PDAC) patients to treat anxiety, anticipatory nausea prior to chemotherapy, and insomnia. We discovered that approximately 1 in 4 pancreatic cancer patients are prescribed BZDs and that significant differences in survival outcomes are associated with the type of BZD prescribed. Lorazepam is associated with poorer recurrence-free survival (HR 3.83 (1.53,9.57)) while alprazolam is associated with improved recurrence-free survival (HR 0.38 (0.16,0.92)). We hypothesize that these differences in survival result from off-target activation of the proton-sensing GPCR, GPR68, by n-unsubstituted BZDs, such as lorazepam. GPR68 is preferentially expressed in PDAC cancer-associated fibroblasts (CAFs). We found that n-unsubstituted BZDs commonly prescribed to PDAC patients potentiate activation of GPR68 in the presence of acidity, subsequently promoting secretion of IL-6 by CAFs in a GPR68-dependent manner. Conversely, we found that n-substituted BZDs, such as alprazolam, decrease IL-6 secretion by CAFs in a GPR68-independent manner. Using a syngeneic KPC transplant model, we found that tumors from mice treated with lorazepam had increased expression of inflammatory signaling pathways and ECM-related genes by RNA sequencing. Histologically, lorazepam-treated tumors had increased levels of collagen, alpha-smooth muscle actin staining, and high levels of ischemic necrosis in the centers of the tumors. Overall, these findings indicate that benzodiazepines can modify the PDAC tumor microenvironment, which may subsequently impact chemotherapeutic efficacy and patient survival. Citation Format: Abigail C. Cornwell, Abdulrahman A. Alahmari, Arwen A. Tisdale, Kathryn Maraszek, Swati Venkat, Michael E. Feigin. Benzodiazepines modify the pancreatic ductal adenocarcinoma tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C048.

Liver metastases 2 years after resection of a very-low-risk duodenal gastrointestinal stromal tumor: a case report

BackgroundGastrointestinal stromal tumors (GISTs) are rare mesenchymal tumors, but are the most common mesenchymal tumors of the gastrointestinal tract. The risk classification of GISTs is based on the tumor size, mitotic index, tumor site, and presence of tumor rupture. Recurrence in the very-low-risk group is extremely rare. We herein report a case of liver metastases 2 years after resection of a very-low-risk duodenal GIST.Case presentationA 57-year-old woman presented to the hospital for evaluation of melena. Esophagogastroduodenoscopy showed bleeding from the exposed blood vessels at the top of a submucosal tumor approximately 20 mm in size located in the second (descending) part of the duodenum, and the bleeding was controlled with electrocoagulation. A GIST was suspected, and the patient underwent wedge resection of the duodenum. The resected specimen contained a 16- × 12-mm (< 20-mm) white submucosal tumor composed of spindle cells with a mitotic count of 4 per 50 high-power fields, and a histologically negative margin was achieved. Immunochemical analysis revealed positive tumor staining for c-kit protein and alpha-smooth muscle actin and negative staining for CD34, desmin, and S-100 protein. Therefore, the tumor was diagnosed as a very-low-risk duodenal GIST based on the Fletcher classification and modified Fletcher classification (Joensuu classification). The postoperative course was uneventful, and the patient was discharged on postoperative day 11. At the follow-up visit 2 years postoperatively, contrast-enhanced computed tomography revealed liver tumors in S8 and S6 measuring 26 × 24 and 10 × 10 mm, respectively. Both lesions showed peripheral dominant hyperenhancement with hypoenhancement inside, indicating tissue degeneration within the tumors. These imaging findings closely resembled those of the duodenal GIST. Hence, the patient was diagnosed with liver metastases of GIST 2 years postoperatively. She was subsequently started on treatment with 400 mg of imatinib. At the time of this writing (2 months after diagnosis), the patient was clinically well and asymptomatic and was continuing imatinib therapy.ConclusionsRecurrence of very-low-risk GISTs is extremely rare. Even a small GIST with low mitotic activity can never be considered completely benign, and long-term follow-up is necessary.

Abstract 3210: Oncostatin M modulates tumor-fibroblast crosstalk in pancreatic cancer without protecting against cachexia

Abstract Cancer cachexia is the involuntary loss of body weight, skeletal muscle, and adipose tissue associated with disease. Cachexia affects 85% of pancreatic cancer patients and patients suffer the greatest average weight loss among solid cancers. Inflammation is a key driver in cancer cachexia, specifically the Interleukin-6/GP130 family of cytokines that activate signaling of the JAK/STAT pathway. Among family members, Interleukin-6 (IL-6) and Leukemia Inhibitory Factor (LIF) promote cachexia and pancreatic cancer; however, less is known of the related Oncostatin M. Here we show cachexia and elevated circulating Osm coincide in the KrasG12D;Trp53R172H;Pdx1:Cre genetically engineered mouse model (KPC-GEMM) of pancreatic cancer and in mice bearing KPC cells implanted orthotopically. Osm expression was elevated in cardiac muscle and adipose tissue in KPC cachexia, while OSM receptor (Osmr) was elevated in cardiac and skeletal muscle and liver, indicating a systemic OSM circuit. Consistent with a feed-forward loop, KPC cells treated with OSM upregulated expression of OSMR. RNA-sequencing of OSM treated KPC and cancer-associated fibroblast (CAF) cell lines revealed activation of inflammation, fibrosis, and tumor progression pathways, including induction of IL-6. Functionally, exogenous OSM caused cardiac dysfunction, local muscle atrophy, fibrosis, and systemic bone loss in wildtype and Il6 null mice, suggesting OSM might mediate cachexia independent of IL-6. We hypothesized that deletion of OSM would be protective in cancer cachexia. Thus 14-week-old WT and Osm null mice were orthotopically implanted with KPC cells or underwent sham surgery; all groups were euthanized when one reached 5% fat mass. KPC tumors caused muscle weakness, body deconditioning and reduced activity as well as anemia and hematological inflammation. These findings were not genotype specific. There were also no differences in KPC-induced cardiac, skeletal muscle or fat wasting, nor in splenomegaly, organ wasting, or tumor size between WT and Osm null mice. Similarly, Osmr null mice showed no differences in KPC cachexia phenotypes versus wildtype mice. Nevertheless, tumors from Osm null mice showed alterations in the stromal compartment, including reduction in collagens I and III, decreased alpha smooth muscle actin staining, and altered mitochondrial metabolism and cellular composition by proteomics and RNAseq. Consistent with a role for OSM in modulating the stroma, excess OSM induced compaction of tumor cells and CAFs in 3D co-cultures. Together these data indicate that OSM signaling influences tumor-fibroblast crosstalk in the pancreatic cancer microenvironment but is not necessary for cachexia in the macroenvironment. Citation Format: Daenique H. Jengelley, Ashok Narasimhan, Joseph E. Rupert, Xiaoling Zhong, Andrew R. Young, Leonidas G. Koniaris, Teresa A. Zimmers. Oncostatin M modulates tumor-fibroblast crosstalk in pancreatic cancer without protecting against cachexia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3210.

Decursin ameliorates carbon-tetrachloride-induced liver fibrosis by facilitating ferroptosis of hepatic stellate cells.

Decursin possesses the potential to alleviate transforming growth factor (TGF)-β-induced hepatic stellate cells (HSCs) activation. However, the mechanisms by which decursin alleviates hepatic fibrosis remain not fully understood. Our aim is to explore the function of decursin on regulating HSCs' activation and hepatic fibrosis. The anti-fibrotic effect of decursin was evaluated by Masson and Sirius red staining, and immunohistochemical (IHC) and quantitative real-time PCR (qRT-PCR) analyses for alpha-smooth muscle actin (α-SMA) and collagen type I(Col1α1) expression. Ferroptosis was assessed by measuring iron concentration, glutathioneperoxidase4 (Gpx4), and prostaglandin endoperoxide synthase 2 (Ptgs2) expression, glutathione (GSH) level, lipid peroxidation, and reactive oxygen species (ROS) level. We found that decursin treatment decreased carbon tetrachloride (CCl4)-induced liver fibrosis. The primary HSCs isolated from decursin-treated group showed an increased Fe2+, lipid ROS level, and decreased Gpx4 and GSH levels compared with HSCs from the model group. Moreover, decursin promoted ferroptosis in activated HSCs in vitro, as evidenced by declined Gpx4 and GSH levels, increased Fe2+, ROS, and Ptgs2 levels compared with control. More important, ferroptosis inhibitor destroyed the anti-fibrosis effect of decursin on HSCs. In summary, these data suggest that decursin has potential to treat hepatic fibrosis.

Hepatocellular Brg1 Promotes CCl4-Induced Liver Inflammation, ECM Accumulation and Fibrosis

Introduction: Brahma-related gene 1 (Brg1) is a catalytic subunit of the SWI/SNF complex and has recently been identified as important for liver regeneration. Liver fibrosis is a progressive pathological process that results in the depletion of the hepatocellular regenerative capacity and ultimately leads to the development of liver cirrhosis and even hepatocellular carcinoma. So far the role of Brg1 in liver fibrosis is unclear. Method: In this study, we examined the effect of Brg1 on the development of liver fibrosis. Hepatocyte-specific Brg1 knockout mice (AlbCre Brg1fl/fl) were injected with CCl4 to induce liver fibrosis. Brg1 expression was determined by Western blot and liver fibrosis was assessed by liver-to-body weight ratio analysis, serum ALT ELISA, Sirius red staining, and alpha-smooth muscle actin staining. Results: Brg1 expression was significantly increased in the fibrotic liver tissue of wild-type mice compared to untreated wild-type mice. The livers of the Brg1 knockout animals showed a significantly reduced liver inflammation, extracellular matrix accumulation and liver fibrosis compared to wild-type mice. Furthermore, we were able to show that HSC activation and inflammatory response during CCl4-induced liver fibrosis are associated with Brg1, which mediates the TNF-α/NF-kB pathway. Conclusions: These results highlight a new aspect of Brg1 in the pathogenesis of liver fibrosis. We have shown that hepatocyte-specific Brg1 deletion prevents liver fibrosis in CCl4-treated mice. In conclusion, Brg1 promotes the progression of liver fibrosis in mice and therefore can be used as a potential therapeutic target for the treatment of patients with liver fibrosis due to chronic injury.

Superimposed tissue formation in human aortic valve disease: differences between stenotic and regurgitant valves

Abstract Introduction The formation of superimposed tissue (SIT), a layer on top of the original valve leaflet, has been described in patients with mitral regurgitation, as a major contributor of valve thickening and probably secondary to increased valve mechanical stress. However, little is known whether SIT formation also occurs in aortic valve disease. Both in the case of aortic stenosis or aortic regurgitation, the aortic valve (AV) is subjected to increased mechanical stresses, although different in type, extent and location. Purpose To characterize SIT formation in aortic stenosis and regurgitation. Methods Human diseased AV leaflets (n=31) were obtained from patients undergoing aortic valve replacement because of aortic stenosis (n=17) or aortic regurgitation (n=14). Histological analysis was performed and elastin staining was used to distinguish the SIT from the original leaflet. Alpha-smooth muscle actin (SMA) staining was performed to identify myofibroblasts and Masson's Trichrome staining to identify collagen fibres. Results In both regurgitant leaflets (RL) and stenotic leaflets (SL) SIT was found at both the ventricular and aortic side (94% of SL, 93% of RL) and could reach up to 50% of total leaflet thickness (Fig. A-C). Although the average SIT thickness did not differ between SL and RL (0.30 mm, standard error of the mean (SEM): ±0.04 for SL vs 0.38 mm, SEM: ±0.05 for RL; p=0.61), the distribution of SIT differed. The SIT at the free edge of the aortic valve was significantly thicker in the RL (0.39 mm, SEM: ±0.06 for SL vs 0.88 mm, SEM: ± 0.07 for RL; p&amp;lt;0.0001), whereas the SIT at the aortic side of the body part was thicker in the SL (0.099 mm, SEM: ±0.023 for SL vs 0.033 mm, SEM: ± 0.021 for RL; p&amp;lt;0.05). Although the SIT comprised of various compositions of extracellular matrix, the overall collagen content was higher in SIT of the SL (212 a.u., SEM: ±4.37 for SL vs 169 a.u., SEM: ±4.06 for RL; p&amp;lt;0.0001). Myofibroblasts were predominantly observed in the SIT as compared to the original leaflet in SL and RL (Fig. D,E; myofibroblast-positive area: 11.6%, SEM: ±3.1 for SIT vs 1.2%, SEM: ±0.3 for original leaflet; p&amp;lt;0.001). The density of myofibroblast in the SIT of the body part of the aortic leaflet, however, was higher in the SL (myofibroblast-positive area: 15.5%, SEM: ±2.0 for SL vs 4.1%, SEM: ±1.3 for RL; p&amp;lt;0.01), whereas the density of myofibroblast in the SIT of the free edge was higher in the RL (myofibroblast-positive area: 15.3%, SEM: ±3.7 for SL vs 4.0%, SEM: ±1.4 for RL; p&amp;lt;0.001). Conclusions Both in aortic stenosis and aortic regurgitation, the AV is characterized by SIT formation but with difference in distribution and composition. These observations suggest the involvement of hemodynamic and mechanical stresses in the regulation of SIT formation of the AV. Understanding the formation of SIT might provide new insights in pathology of AV disease. Funding Acknowledgement Type of funding sources: None.

Laparoscopic and endoscopic cooperative surgery for leiomyosarcoma of the stomach: a case report with a review of the literature

BackgroundLeiomyosarcoma is a rare tumor that could originate from the gastrointestinal tract, uterus, kidney, retroperitoneum, and the soft tissues of the extremities. It accounts for only 1% of all gastrointestinal mesenchymal tumors and primary leiomyosarcoma of the stomach is extremely rare. Most cases reported as leiomyosarcoma of the stomach before the development of KIT immunohistochemistry might be gastrointestinal stromal tumors (GISTs) of the stomach and only 18 cases of leiomyosarcoma of the stomach have been reported since early 2000s. We report here a patient with leiomyosarcoma of the stomach treated by laparoscopic and endoscopic cooperative surgery (LECS).Case presentationA 59-year-old man was referred to our hospital for an early gastric cancer, which was initially treated by endoscopic submucosal dissection. Six months after his initial treatment, a follow-up esophagogastroduodenoscopy revealed a small polypoid lesion at the lesser curvature of the proximal stomach, which appeared to be a hyperplastic polyp. However, one and a half years later, the lesion grew and showed more irregular surface. Biopsy at the time revealed smooth muscle cell proliferation suggestive of leiomyoma. Three years later, the lesion grew even larger and biopsy showed pleomorphic spindle cells. Immunohistochemical study showed positive staining for alpha-smooth muscle actin and desmin, but negative for c-kit and CD34. Ki-67 labeling index was nearly 60%. Based on these findings, the diagnosis of leiomyosarcoma was established. The patient subsequently underwent a partial gastrectomy by LECS. The patient is currently in good condition without recurrence or metastasis at 12 months after surgery.ConclusionsLeiomyosarcoma of the stomach is extremely rare. This is the first report of leiomyosarcoma of the stomach treated by LECS. We could also follow its appearance change through endoscopic examination for 3 years.

Hawthorn Herbal Preparation from Crataegus oxyacantha Attenuates In Vivo Carbon Tetrachloride -Induced Hepatic Fibrosis via Modulating Oxidative Stress and Inflammation.

Hawthorn (HAW) is a herbal preparation extracted from Crataegus oxyacantha. HAW has cardioprotective, antioxidants, anti-inflammatory, and anti-hypotensive effects. HAW’s effect on hepatic fibrosis remains, however, unknown. This study evaluated the impact of HAW on carbon tetrachloride (CCl4)-induced hepatic fibrosis in rats and elucidated its mechanisms. HAW reduced liver index and the serum liver enzyme markers and reduced liver damage, and fibrosis as confirmed by histopathological scoring of hematoxylin-eosin staining. Collagen deposition was reduced in HAW group compared to CCl4 group as confirmed by Masson staining, hydroxyproline content, and both mRNA and protein levels of alpha-smooth muscle actin, collagen 1 and 3. HAW also down regulated the gene expressions of inflammatory markers including interleukin-IL-1β, tumor necrosis factor-α, transforming growth factor-β 1, nuclear factor kappa-B, and cyclooxygenase-2 and decreased the myeloperoxidase activity. The effects of HAW was also associated with decreased levels of hepatic oxidative stress markers (malondialdehyde and P.Carbonyl) and with increased activity of superoxide dismutase. Those effects are possibly mediated by blocking the pro-oxidant machinery and down regulating the inflammatory and profibrotic responses. Finally, chlorogenic acid, epicatechin, rutin, vitexin quercetin, and iso quercetin were identified as the major species of polyphenols of the HAW herbal preparation used here. Therefore, HAW’s potent protecting effects against liver fibrosis predicts a significant beneficial application.

Evolution of ischemia and neovascularization in a murine model of full thickness human wound healing.

Translation of wound healing research is limited by the lack of an appropriate animal model, due to the anatomic and wound healing differences in animals and humans. Here, we characterize healing of grafted, full-thickness human skin in an in vivo model of wound healing. Full-thickness human skin, obtained from reconstructive operations, was grafted onto the dorsal flank of NOD.Cg-KitW41J Tyr + Prkdcscid Il2rgtm1Wjl /ThomJ mice. The xenografts were harvested 1 to 12 weeks after grafting, and histologic analyses were completed for viability, neovascularization, and hypoxia. Visual inspection of the xenograft shows drying and sloughing of the epidermis starting at week four. By week 12, the xenograft appears healed but has lost 63.05 ± 0.24% of the initial graft size. There is histologic evidence of epidermolysis as early as 2 weeks, which progresses until week 4, when new epidermis appears from the wound edges. Epidermal regeneration is complete by week 12, although the epidermis appears hypertrophied. An initial increase of infiltrating immune mouse cells into the xenograft normalizes to baseline 6 months after grafting. Neovascularization, as evidenced by positive staining for the proteins human CD31 and alpha smooth muscle actin, is present as early as 2 weeks after grafting at the interface between the xenograft and the mouse tissue. CD31 and alpha smooth muscle actin staining increased throughout the xenograft over the 12 weeks, leading to greater viability of the tissue. Likewise, there is increased Hypoxia Inducible Factor 1-alpha expression at the interface of viable and nonviable tissue, which suggest a hypoxia-driven process causing early graft loss. These findings illustrate human skin wound healing in an ischemic environment, providing a timeline for use of full thickness human skin after grafting in a murine model to study mechanisms underlying human skin wound healing.

Effect and mechanism of miR-223 on fibrosis-related signaling pathway in rat cardiomyocytes

Objective: To investigate the protective effect of microRNA 223 (miR-223) on cardiac fibrosis-related signaling pathway and its regulation on expression of Twist family basic helix-loop-helix transcription factor 1 (Twist1) and transforming growth factor-β1 receptor 2 (TGFBR2) in rat cardiomyocytes. Methods: Rat cardiomyocytes (H9c2) were cultured in vitro and treated with TGF-β to induce myocardial fibrosis. The miR-223 group was transfected with miR-223 lentivirus and miR-223-NC group was transfected with miR-223-NC lentivirus. Model group and blank control group had no transfection. Immunocytochemistry staining of alpha-smooth muscle actin (α-SMA) was used to calculate myocardial fibrosis. The mRNA level of miR-223, collagen Ⅰ, collagen Ⅲ, Twist1 and TGFBR2 were detected by real-time PCR. The protein level of Twist1, TGFBR2, collagen Ⅰ, collagen Ⅲ and α-SMA were detected by Western blot. Target regulation of miR-223 on Twist1 and TGFBR2 3'UTR was verified by double luciferase reporter gene system. Results: The average optical density of α-SMA-positive cardiomyocytes in miR-223 group (0.089±0.013) was significantly lower than that in model group and miR-223-NC group (0.134±0.018, 0.132±0.016, respectively). The mRNA level of collagen Ⅰ, collagen Ⅲ, Twist1 and TGFBR2 in miR-223 group were significantly lower than that in model group and miR-223-NC group (all P<0.05). The protein level of Twist1, TGFBR2, collagen Ⅰ, collagen Ⅲ and α-SMA in miR-223 group was significantly lower than model group and miR-223-NC group (all P<0.05). Twist1, TGFBR2 3'UTR wild-type double luciferase reporter plasmids and miR-223 mimics were co-transfected in 293T cells, and luciferase activity was significantly reduced (0.48±0.06 vs 0.92±0.17 and 0.51±0.07 vs 0.94±0.12). Conclusion: MiR-223 may inhibit the activation of fibrosis-related signaling pathway in cardiomyocytes by down-regulating the expression of Twist1 andTGFBR2 genes.

Cancer-associated fibroblasts promote the migration and invasion of gastric cancer cells via activating IL-17a/JAK2/STAT3 signaling.

Cancer-associated fibroblasts (CAFs), as the activated stroma cells, contribute to tumor progression via the release of cytokines, growth factors, and hormones. However, neither the factors produced by CAFs nor the molecular mechanisms were illuminated very well in gastric cancer (GC). Immunohistochemical staining of alpha-smooth muscle actin (α-SMA) was applied to examine the number of CAFs in GC samples from 227 patients. ELISA and qRT-PCR were performed to detect the expression of interleukin 17a (IL-17a). The migration and invasion of GC cells were determined by the Transwell assay. The expressions of JAK2, STAT3, MMP-2, MMP-9, TIMP-1, and TIMP-2 were measured by western blotting. IL-17a was blocked with a polyclonal antibody, and JAK2/STAT3 signaling pathway was blocked by a specific inhibitor AG490. High CAFs in GC tissues were positively correlated with advanced TNM stage and perineural invasion. Furthermore, GC patients with high CAFs in tumor tissues had an obvious worse disease-free survival (DFS) and disease-special survival (DSS). Multivariate analysis showed that high CAFs in GC tissues were an independent risk factor for DFS and DSS. CAFs expressed IL-17a significantly after GC cell co-culture. CAFs markedly enhanced the migration and invasion abilities of AGS and SGC-7901 cells. Moreover, CAFs co-culture resulted in increased levels of MMP2/9, reduced expressions of TIMP1/2, and activation of the JAK2/STAT3 signaling pathway in the GC cells. IL-17a neutralizing antibody or JAK2 inhibitor AG490 can significantly inhibit the effects of CAFs on the migration, invasion, MMP2/9, TIMP1/2, and JAK2/STAT3 pathways of GC cells. CAFs correlated with unfavorable clinical features and poor prognosis of GC patients. CAFs secreted IL-17a, which promoted the migration and invasion of GC cells through activating JAK2/STAT3 signaling. These results may identify IL-17a as a promising prognostic marker and therapeutic target of GC.

Evaluation of Cancer-Associated Fibroblasts Markers in Hepatocellular Carcinoma

Abstract Background: Cancer associated fibroblasts (CAFs) are main components of tumor microenvirnoment. They act an essential role in tumor progression, invasion and unrespon-siveness to chemotherapy. Aim of Study: This work aims to study the relation of CAFs with hepatocellular carcinoma (HCC) developement and progression. Material and Methods: This is an uncontrolled retrospec-tive study carried upon 30 cases of HCC. Cases were collected from archives of Pathology Department and Early Cancer Detection Unit (ECDU), Faculty of Medicine, Benha Univer-sity, and from International Medical Center during the years 2010-2015. Podoplanin (D2-40), alpha smooth muscle actin (a-SMA) and CD31 immunohistochemichal staining were performed and evaluated for each case. Results: Both D2-40 and a-SMA showed different distri-butions and levels of expression. D2-40 positivity was localized to the peri-tumoral area, while a-SMA was detected in both intra- and peritumoral areas. Expression of both markers was not statistically related to each other (p>0.05). D2-40 was positive in 73.3% of casesand a-SMA showed high expres-sionin 60% of cases. Both positive D2-40 and high a-SMA showed positive relations to tumor grade (p

Interrogating the Spatial Architecture of Human Bone Marrow Via Imaging Mass Cytometry

Hematopoietic stem cell (HSC) proliferation, self-renewal, differentiation, and trafficking are dependent, in part, upon signals generated by stromal cells in the bone marrow. Stromal cells are organized into niches that support specific subsets of hematopoietic progenitors. Intimate interactions between HSCs and neighboring stromal cells coordinate hematopoietic responses during periods of physiologic stress, while also maintaining the lifelong integrity of the hematopoietic stem cell pool. Hematopoietic niches are comprised of a heterogeneous population of stromal and hematopoietic cells. The identity and function of the known stromal cell subsets have primarily been gleaned through genetic manipulation of mouse models. In humans, the spatial organization of these stromal cells in bone marrow and the signals they generate to regulate hematopoiesis are poorly understood. Current methods to characterize bone marrow mesenchymal stromal cells in humans include: 1) immunostaining of bone sections; 2) analysis of flow sorted stromal cells; and 3) analysis of ex vivoexpanded mesenchymal stem/progenitors. Major limitations to all of these approaches exist that relate to the heterogeneity of bone marrow stromal cells, the lack of markers that reliably distinguish different stromal cell populations, and inherent technical limitations of the assays, such as the number of markers that can be analyzed at one time. Here we report our efforts to use imaging mass cytometry imaging (IMC) to interrogate the complex cellular architecture of human bone marrow. IMC allows for the simultaneous detection of up to 40 markers through the use of antibodies conjugated to elemental metal tags acquired by time-of-flight mass spectrometry. We first used standard immunostaining techniques to develop a panel of antibodies compatible with archived formalin-fixed paraffin-embedded (FFPE) human bone marrow specimens using a heat-induced epitope retrieval method. Hematopoietic lineages have been successfully identified using CD11b, CD68, CD15, CD14, CD16, CD11c, CD20, CD3, CD4, CD8a, CD38, CD45RA, CD45RO, CD235a, CD71, CD34, CD31; stromal cells and structures with CXCL12, alpha-smooth muscle actin, collagen I, vimentin; and nuclear staining using Ki67, Histone H3, and DNA intercalator. We used panels consisting of up to 18 of these antibodies each to test IMC on FFPE human bone marrow specimens. Imaging was performed using the Hyperion Imaging System (Fluidigm), which consists of a UV laser scanning module to capture regions of more than 10,000 cells with 1-micron resolution coupled with a Helios Mass Cytometer. Image analysis was performed by creation of a cell segmentation mask using CellProfiler software and high dimensional analysis in histoCAT. Representative images and corresponding dimensional reduction with t-SNE are shown in Figure 1A-H, demonstrating successful discrimination of distinct hematopoietic lineages. Gating on subpopulations within the t-SNE clusters can be projected on to the original image to illustrate spatial distribution and marker co-expression, as an example CD3+ and CD8+ cells are shown in Figure 1I. These data indicate that IMC allows for highly multiplexed analysis of bone marrow cell populations. Developing imaging techniques for analysis of tissue-banked FFPE bone marrow samples would have broad applications for translational research on hematologic diseases. In particular, this technology has tremendous potential to advance understanding of the spatial architecture of human bone marrow and to investigate alterations in the bone marrow environment in malignant hematopoiesis. Disclosures Oh: Incyte: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

Anti-atherosclerotic Effects of Oats (Avena Sativa) on Blood Vessels of Albino Rats’ Tongue

Objective: To investigate anti-atherosclerotic effect of oats against increased fat diet which result in atherosclerosis in albino rats. Methodology: 30 adult male albino rats divided into three groups, 10 rats each: group I: negative control take basal diet, group II take diet with increased cholesterol level for 8 weeks, group III fed on diet with increased cholesterol level for 60 days then given oats-supplemented diet (20% b/w) besides the same diet of cholesterol for another 4 weeks. The animals were euthanized under mild ether anesthesia at the end of 8th week for group II and at the end of 12th week for group III. and the blood was taken from inner canthus of the eye, total serum cholesterol level has been observed and each animal has cholesterol level more than 250 mg/dl was estimated to be hyperlipidemic rat. Rats’ tongue carefully dissected out and processed for Haematoxylin & Eosin and alpha smooth muscle actin staining. Results: histological examination revealed that atherosclerosis would lead to endothelial damage of the blood vessels of the tongue; rats received oats in their diet showed regeneration of the endothelial cells. Immuno-histochemical study showed increase positive immunoreactivity in blood vessels of tongue of group II rats that received high fat diet compared with rats of negative control group or rats of oat groupConclusion: including oats in diet can be effective in reducing serum cholesterol.

Complete Myogenic Differentiation of Adipogenic Stem Cells Requires Both Biochemical and Mechanical Stimulation.

Vascular tissue engineering of the middle layer of natural arteries requires contractile smooth muscle cells (SMC) which can be differentiated from adipose-derived mesenchymal stem cells (ASC) by treatment with transforming growth factor-β, sphingosylphosphorylcholine and bone morphogenetic protein-4 (TSB). Since mechanical stimulation may support or replace TSB-driven differentiation, we investigated its effect plus TSB-treatment on SMC orientation and contractile protein expression. Tubular fibrin scaffolds with incorporated ASC or pre-differentiated SMC were exposed to pulsatile perfusion for 10days with or without TSB. Statically incubated scaffolds served as controls. Pulsatile incubation resulted in collagen-I expression and orientation of either cell type circumferentially around the lumen as shown by alpha smooth muscle actin (αSMA), calponin and smoothelin staining as early, intermediate and late marker proteins. Semi-quantitative Westernblot analyses revealed strongly increased αSMA and calponin expression by either pulsatile (12.48-fold; p < 0.01 and 38.15-fold; p = 0.07) or static incubation plus TSB pre-treatment (8.91-fold; p < 0.05 and 37.69-fold; p < 0.05). In contrast, contractility and smoothelin expression required both mechanical and TSB stimulation since it was 2.57-fold increased (p < 0.05) only by combining pulsatile perfusion and TSB. Moreover, pre-differentiation of ASC prior to pulsatile perfusion was not necessary since it could not further increase the expression level of any marker.

Biobanking of Dehydrated Human Donor Corneal Stroma to Increase the Supply of Anterior Lamellar Grafts.

To investigate the effect of dehydration on human donor corneal stroma for biobanking. Epithelium and endothelium of research-grade human donor corneas (n = 12) were scraped off, leaving a bare stroma with attached sclera. The tissues were placed in a large Petri dish prefilled with silica gel in the periphery and stored at room temperature for 14 days. At the end of preservation, the tissues were rehydrated by being submerged in phosphate-buffered saline for 15 minutes. Transparency (using a custom-built device) and thickness (using optical coherence tomography) measurements were recorded before dehydration, after dehydration, and after rehydration of the tissues. Periodic acid-Schiff and alpha-smooth muscle actin (α-SMA) staining before dehydration and after rehydration were performed to determine the presence of keratocytes and expression of α-SMA. Tensile stress-strain before dehydration and after rehydration was performed to evaluate the biomechanical properties. No difference in corneal transparency before dehydration (69.57 ± 6.41%) and after rehydration (67.37 ± 2.82%), P = 0.36, was observed. The corneas were more compact after dehydration. A significant change in thickness between before dehydration (625.8 ± 75.58 μm) and after rehydration (563.6 ± 15.77 μm) stage, P = 0.03, was noticed. The thickness was reduced to 147.6 ± 3.71 μm when dehydrated. Periodic acid-Schiff staining showed presence of stromal keratocytes and α-SMA protein expressed in control, dehydrated, and rehydrated corneas. There was no significant difference in the stiffness between control (27.86 ± 11.65 MPa) and rehydrated corneas (31.46 ± 11.41 MPa). Human donor corneal stroma can be biobanked for up to 2 weeks in a dehydrated condition without losing their molecular or biomechanical properties after rehydration.