MMP-9 Staining Research Articles

AnGong NiuHuang (AGNH) pill attenuated traumatic brain injury through regulating NF-κB/Nlrp3 axis and glycerophospholipid metabolism

BackgroundTraumatic brain injury (TBI), especially neuroinflammation after TBI persists for a long time and causes significant neurodegenerative pathologies and neuropsychiatric problems. PurposeIn this study, the neuroprotective effect of AnGong NiuHuang (AGNH) on TBI was investigated and the mechanism was revealed by integrating multiple omics. MethodsThe rats with TBI were administrated with AGNH for 5 consecutive days and the effect was evaluated by using modified neurologic severity score (mNSS), brain edema, H&E staining, Nissl staining and TUNEL staining. The mechanism was revealed by using RNA sequencing (RNA-seq) and metabolomic analysis. The inflammatory factors, apoptosis-related proteins and identified vital targets were validated by enzyme-linked immunosorbent assay, western blotting and immunofluorescence staining. ResultsAdministration of AGNH decreased mNSS, brain edema, brain structure damage, but increased Nissl body density in the rats with TBI. Additionally, AGNH reduced IL-1β, IL-17A, TNF-α, MMP9, MCP-1, IL-6, Bax and TUNEL staining,but elevated Bcl2 level. Integrating transcriptomic analysis and metabolomic analysis identified vital targets and critical metabolic pathways. Importantly, AGNH treatment reduced the expression of TLR4, MYD88, NLRP3, BTK, IL-18 and Caspase 1 as well as glycerophospholipid metabolism-related protein AGPAT2 and PLA2G2D, and decreased the nuclear translocation of NF-κB p65 in the brain of TBI rats. Additionally, AGNH increased phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylethanolamine (PE), but decreased 1-acyl-sn-glycero-3-phosphocholine (LysoPC) in the metabolic pathway of glycerophospholipid metabolism. ConclusionTaken together, AGNH inhibited NF-κB/NLRP3 axis to suppress neuroinflammation, cell apoptosis and pyroptosis, and improved metabolic pathways of glycerophospholipid metabolism after TBI.

Evaluation of MMP-9, MMP-13, MMP-21, and TIMP-1 expressions in malign melanom, dysplastic nevi, and banal nevi.

Although the role of MMPs in the pathogenesis of melanoma is known, few studies have investigated their role in the development of nevi and dysplastic nevi. This study aims to search the expression differences of MMP-9, MMP-13, MMP-21, and TIMP-1 between malignant melanoma (MM), intradermal nevi (IDN), and dysplastic nevi (DN). MMP-9, MMP-13, MMP-21, and TIMP-1 antibodies were studied immunohistochemically for 60 cases in our pathology clinic archive between 2013 and 2014. The MM group had the highest expression percentage and intensity for MMP-9 (p<0.001). There was no statistical significance between MMP-13 expression intensities of lesion cells and stromal cells and stromal expression intensities (p>0.05). MMP-21 lesion staining intensities in DN and MM compared to IDN were statistically significant (p=0.001, p=0.011, respectively). For TIMP-1, there was a significant difference between the IDN and the MM group regarding the staining proportion of lesion cells (p<0.01). There was a statistically significant difference in all groups according to lesion cells' expression intensity. (IDN-DN p<0.001, IDN-MM p=0.044, DN-MM p<0.001). The following markers can be helpful when lesions cannot be differentiated; increased staining proportions and intensity of MMP-9 in both lesion and stromal cells favor MM in cases where MM and IDN cannot be differentiated. The increased MMP-13 staining proportion of lesion cells can favor DN in cases where the pathologist cannot differentiate DN and MM. Intense expression of MMP-21 by lesion cells can be a potential marker for evaluating the lesion in favor of DN in cases where DN and IDN cannot be differentiated. The high expression intensity of TIMP-1 in lesion cells can favor DN in cases where there is ambiguity between DN and MM. High expression proportion and intensity of stromal cells of TIMP-1 can be useable in favor of MM in cases where MM and DN cannot be differentiated.

Metalloproteinase 9 immunostaining profile is positively correlated with tumor grade, extraprostatic extension and biochemical recurrence in prostate cancer

Metastasis is the main problem in the treatment of prostate cancer (PCa), and for it to occur, proteolytic enzymes must remodel the extracellular matrix (ECM) surrounding the tumor. The most important group of enzymes with this action include the matrix metalloproteinases (MMPs), which act on various substrates cleaving ECM components. The present study aimed to evaluate the protein immunostaining profiles of matrix metalloproteinase 2 (MMP-2) and 9 (MMP-9) in PCa Brazilian patients using the indirect immunohistochemical methodology. The tissue samples (n = 178), 60 from malignant tumor, 58 from adjacent non-tumor, and 60 from ECM, were evaluated according to the immunostaining intensity. The malignant tumor cytoplasmic MMP-2 immunostaining was more intense than in ECM (p = 0.001), but it did not correlate with any clinical-pathological parameter. The MMP-9 staining was similar in tumor cytoplasm, adjacent non-tumor cytoplasm and ECM, but showed significant positive correlations with ISUP grade (p = 0.044; Tau=0.249), extraprostatic extension (p = 0.025; Tau=0.309), and biochemical recurrence (p = 0.048; Tau=0.306). A significant positive correlation was also observed between MMP-2 and MMP-9 in all cell compartments analyzed. Although further research is warranted to elucidate the precise mechanisms underlying these observations, our findings suggest MMP-9 as a promising candidate marker for tissue invasion that could be used in predicting the progression and prognosis of PCa.

Immunohistochemical Features of MMP-9 and pSTAT1 in Granuloma Annulare and Sarcoidosis: A Comparative Study of 62 Cases.

Granuloma annulare (GA) and sarcoidosis are granulomatous inflammatory diseases that share similarities. To identify the histological and immunohistochemical (IHC) features of GA and sarcoidosis. A retrospective review of 36 patients with GA and 26 with sarcoidosis was performed. Results from hematoxylin and eosin (H&E) staining and IHC staining of MMP-9 and pSTAT1 within the skin lesions of GA and sarcoidosis were analyzed, and random forest was applied for developing a predictive model. Significantly greater expressions of MMP-9 (especially in elastic fibers, EFs, P < 0.0001) and pSTAT1 (P = 0.0003) were observed in lesion samples of GA versus sarcoidosis patients. In GA patients, MMP-9 was significantly upregulated in the interstitial type (P = 0.0222), while staining of pSTAT1 was positively correlated with the area of mucinous collagen in palisading GA (R = 0.5356, P = 0.0484). In sarcoidosis patients, MMP-9 (R = -0.7127, P = 0.0009) and pSTAT1 (R = -0.5604, P = 0.0067) were found to show stronger expressions in lesions with less lymphocyte infiltration. The predictive model demonstrated an AUC of 0.9675. These results indicate that MMP-9 and pSTAT1 might exert roles in granulomatous inflammation in different modes, and the presence of more robust MMP-9 staining in EFs appears to be more suggestive of GA.

Targeting the Tumor Immune Microenvironment Could Become a Potential Therapeutic Modality for Aggressive Pituitary Adenoma

This study aimed to explore the relationship between the aggressiveness and immune cell infiltration in pituitary adenoma (PA) and to provide the basis for immuno-targeting therapies. One hundred and three patients with PA who underwent surgery at a single institution were retrospectively identified. The infiltration of macrophages and T-lymphocytes was quantitatively assessed. The number of CD68+ macrophages was positively correlated with Knosp (p = 0.003) and MMP-9 expression grades (p = 0.00). The infiltration of CD163+ macrophages differed among Knosp (p = 0.022) and MMP-9 grades (p = 0.04). CD8+ tumor-infiltrating lymphocytes (TILs) were also positively associated with Knosp (p = 0.002) and MMP-9 grades (p = 0.01). Interestingly, MGMT expression was positively correlated with MMP-9 staining extent (p = 0.000). The quantities of CD8+ TILs (p = 0.016), CD68+ macrophages (p = 0.000), and CD163+ macrophages (p = 0.043) were negatively associated with MGMT expression levels. The number of CD68+ macrophages in the PD-L1 negative group was significantly more than that in the PD-L1 positive group (p = 0.01). The rate of PD-L1 positivity was positively correlated with the Ki-67 index (p = 0.046) and p53 expression (p = 0.029). Targeted therapy for macrophages and CD8+ TILs could be a helpful treatment in the future for aggressive PA. Anti-PD-L1 therapy may better respond to PAs with higher Ki-67 and p53 expression and more infiltrating CD68+ macrophages. Multiple treatment modalities, especially combined with immunotherapy could become a novel therapeutic strategy for aggressive PA.

Safety and Performance of an Artificial Tear Containing Arabinogalactan, Trehalose and Hyaluronic Acid in the Treatment of Dry Eye in Subjects with Signs of Inflammation of the Ocular Surface

Background: In this study it has been evaluated the safety and the clinical performance of a new combination of topical solution based on arabinogalactan, trehalose and hyaluronic acid, in reducing symptoms and signs of inflammation in a selected population affected by dry eye disease. Methods: The study enrolled 25 patients (ranging from 57 to 71 years) with signs and symptoms of dry eye disease. They received a combination therapy with arabinogalactan, trehalose and hyaluronic acid one drop three times a day for the first week and subsequently according to their symptoms. Ocular surface disease index score, tolerability and adherence were used to evaluate subjective symptoms of dry eye disease at enrolment (T0), week 1 (T1) and week 4 (T2). Clinical signs were assessed at T0 and T2. The assessment plan included: slit-lamp examination of the anterior segment, uncorrected and corrected visual acuity, ocular protection index, tear film break up time, osmolarity, meniscometry, evaluation of conjunctival hyperemia according to Efron’s classification, the presence of MMP-9, conjunctival and corneal staining and intraocular pressure. Results: The study group showed significant improvement in several clinical parameters after treatment. Ocular surface disease index score was significantly lower at the end of the follow up period, also evaluation of tolerability (ocular burning, ocular itching and blurred vision), before and after instillation, showed a statistically significant improvement at T0 and T1. Improvements in tear film break up time, meniscometry results and conjunctival and corneal hyperemia were statistically significant in the study group (p &lt;0.05). A statistically significant, decrease in MMP-9 levels was also observed. Conclusions: Preliminary findings suggest that the new combination of topical solution based on arabinogalactan, trehalose and hyaluronic acid, could be effective in reducing early inflammatory markers, symptoms and signs, associated with the progression of dry eye disease.

Association between the Expression of KISS1, KISS1R and MMP-9 in Invasive Breast Carcinomas

Metastases are the leading cause of fatal outcome in patients with breast cancer, therefore different proteins related to metastasis are widely studied for the potential to control the spread of cancer cells. It has been shown in various types of cancers that the anti-metastatic role of the KISS1/KISS1R system has been performed by suppressing metalloproteinase 9 (MMP-9) expression, but there is evidence that the relationship between KISS1/KISS1R signalling and MMP-9 in breast cancer seems to be different. The aim of the current study was to assess the association between protein expression of KISS1, KISS1R and MMP-9 using software-based measurement of staining intensity and the possible link to tumour size, grade and receptor status in invasive breast carcinomas. Standard histopathological parameters were determined by an experienced pathologist and immunohistochemical staining for KISS1, KISS1R and MMP-9 was performed on tissue samples from 54 patients. Evaluation of staining intensity was performed using ImageJ software on RGB images by two operators. Results showed significant strong positive correlation between KISS1, KISS1R and MMP-9 and strong expression of studied proteins in tumour associated stromal cells. No association was found between the expression of the studied proteins and size, receptor status or grade. Higher expression of KISS1R, however, was observed in HER2-negative compared to HER2-positive carcinomas, which might indicate an alternative pathway for stimulating pro- proliferation of tumour cells when HER2 expression is low.

Matrix metalloproteinase 2 is a target of the RAN-GTP pathway and mediates migration, invasion and metastasis in human breast cancer

RAS-related nuclear protein(RAN) is a nuclear shuttle and normally regulates events in the cell cycle. When overexpressed in cultured cells, it causes increases in cell migration/invasion in vitro and its overexpression is associated with early breast cancer patient deaths in vivo. However, the underlying mechanism is unknown. The effect of RAN overexpression on potential targets MMP2, ATF3, CXCR3 was investigated by Real-Time PCR/Western blots in the triple receptor negative breast cancer(TRNBC) cell line MDA-MB231 and consequent biological effects were measured by cell adhesion, cell migration and cell invasion assays. Results showed that knockdown of RAN lead to a reduction of MMP2 and its potential regulators ATF3 and CXCR3. Moreover, knockdown of ATF3 or CXCR3 downregulated MMP2 without affecting RAN, indicating that RAN regulates MMP2 through ATF3 and CXCR3. Knockdown of RAN and MMP2 reduced cell adhesion, cell migration and cell growth in agar, whilst overexpression of MMP2 reversed the knockdown of RAN. Furthermore, immunohistochemical staining for RAN and MMP2 are positively associated with each other in the same tumour and separately with patient survival times in breast cancer specimens, suggesting that a high level of RAN may be a pre-requisite for MMP2 overexpression and metastasis. Moreover, positive immunohistochemical staining for both RAN and MMP-2 reduces further patient survival times over that for either protein separately. Our results suggest that MMP2 expression can stratify progression of breast cancers with a high and low incidence of RAN, both RAN and MMP2 in combination can be used for a more accurate patient prognosis. Simple summaryRan is an important regulator of normal cell growth and behaviour. We have established in cell line models of breast cancer (BC) a molecular pathway between RAN and its protein-degrading effector MMP-2 and properties related to metastasis in culture. Using immunohistochemistry (IHC) staining of primary BCs, we have shown that RAN and MMP-2 are on their own significantly associated with patient demise from metastatic BC. Moreover, when staining for MMP-2 is added to that for RAN in the primary tumours, there is a significant decrease in patient survival time over that for either protein alone. Thus a combination of staining for RAN and MMP2 is an excellent marker for poor prognosis in breast cancer.

CXCR4 and MMP-9 Immunoexpression are Associated with Metastasis in Infiltrating Urothelial Carcinoma of The Bladder

Background: Infiltrating urothelial carcinoma of the bladder (IUCB) is an aggressive urinary tract cancer. Most of the patients were diagnosed with advanced stages, and many have metastasized. Metastasis is responsible for 90% of cancer deaths compared to the primary tumor itself. In previous research, the expression of CXCR4 and MMP-9 has been suggested to correlate with metastasis and prognosis in several cancers. This study aimed to analyze the association of CXCR4 and MMP-9 expression with metastasis in infiltrating urothelial carcinoma of the bladder. Methods: This cross-sectional study included 40 selected paraffin-embedded tissue blocks from histopathologically diagnosed IUCB patients, consisting of 20 cases of non-metastasizing and 20 cases of metastasizing IUCB. Immunohistochemical staining for CXCR4 and MMP-9 was performed in all cases. All data were analyzed using the Chi-Square test with p &lt; 0.05 of a significant level and then processed using SPSS 24.0 for Windows.Results: High CXCR4 immunoexpression was found in 18 (45%) of all cases, and high MMP-9 immunoexpression was found in 19 (47.5%) cases. High immunoexpression of CXCR4 and MMP9 in infiltrating urothelial carcinoma of the bladder showed significant association with metastasis (p &lt; 0.05). The stepwise logistic regression analysis revealed that both CXCR-4 and MMP-9 immunoexpressions were independent prognostic factors for metastasis.Conclusions: CXCR4 and MMP-9 play a role in the metastatic process in infiltrating urothelial carcinoma of the bladder.

Expression of matrix metalloproteinase-2 and survivin in pancreatic carcinoma

Background and Aim Matrix metalloproteinase (MMP) is a member of the MMP family, which is a zinc-dependent endopeptidase that degrades all extracellular matrix components and vascular basement membrane. Survivin plays a leading role in the process of apoptosis regulation. Survivin is unique for its expression in human malignancies but not in normal adult cells. This study aimed to assess immunoexpression of MMP-2 and survivin in pancreatic carcinoma. Patients and methods A total of 25 pancreatic carcinomas were evaluated for immunohistochemical expression of MMP-2 and survivin. The expression was evaluated by immunoreactive score that combines the intensity of immunoreactions with the percentage of positive cells. Results In this study, 24/25 of the cases were pancreatic ductal adenocarcinoma (PDAC) and 1/25 was acinar cell carcinoma. Regarding tumor differentiation, PDAC included five cases of adenocarcinoma that were well differentiated, 16 cases were moderately differentiated, and three cases were poorly differentiated. Pancreatic carcinoma specimens showed positive cytoplasmic MMP-2 staining with high expression in 22/25 (88%) studied specimens ranging from 6/9 to 9/9 with high extent and intensity. Survivin staining with high expression was seen in 23/25 (92%) studied specimens, ranging from 9/12 to 12/12 with high extent and intensity. Conclusion MMP-2 and survivin expressions were positive in pancreatic carcinoma with various grades but negative in non-neoplastic parenchymal, acinar, and pancreatic ductal epithelium. There were significant positive relationships between MMP-2 and survivin expression and poor histological grade in PDAC (P&lt;0.024 and 0.021, respectively).

Abstract P2-08-16: The cross-talk between nuclear p-ser294-FOXO3a and cytoplasmic MMP-2 in breast cancer survival and treatment response

Abstract Background The forkhead protein FOXO3a, the member of the forkhead box O (FOXO) transcription factors, controls a wide spectrum of carcinogenesis, therapy response and progression in human cancer. Emerging evidence suggest that phosphorylation of FOXO3a by ERK and p38 at Serine 294 induces its nuclear localization or exclusion to the cytosol, therefore regulating FOXO3a transcriptional activity and consequently promoting cell proliferation and tumorigenesis. Several proteins regulated by FOXO3a, such as MMP-2,9 and 13, are involved in metastasis. We aim to explore the potential role of p-ser294-FOXO3a and MMPs, and interplay between both in breast cancer. Materials and Methods Paraffin-embedded tissue microarrays from 350 breast cancer patients were enrolled. Immunohistochemical analysis was performed, and the expression patterns of p-ser294-FOXO3a, MMP-2, MMP-9, MMP-13 were categorized by semiquantitative method and further correlated with the clinicopathological parameters using the x2 test. Survival curves were calculated using the Kaplan-Meier method, while the significance was determined by log rank test. The biological impacts and underlying mechanisms of p-ser294-FOXO3a were analyzed by transwell assay, RT-PCR, western blotting immunofluorescence staining. co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) assay in breast cancer cells upon transfection with FOXO3a and FOXO3a mutant (S294A). Results Our results showed that nuclear p-Ser294-FOXO3a index was inversely correlated with tumor grade (p=0.011) and relapse (p=0.053), but positively correlated only with cytoplasmic MMP-2 intensity (p=0.010). It is noted that cytoplasmic MMP-2 intensity was positively correlated with tumor grade (p=0.032) and size (p=0.028). Intriguingly, decreased nuclear p-Ser294-FOXO3a index was significantly associated with an improved overall survival rate (p=0.004) and of those patients bearing high MMP-2 staining (p&amp;lt;0.001) or negative ER status (p=0.006), and those receiving hormone therapy (HT) (p=0.030) or chemotherapy (p=0.001). Ectopic FOXO3a overexpression increased invasion rate which was inhibited by tamoxifen (TAM) in only ER-positive MCF-7 cells. In addition, TAM inhibited the invasion rates of ERα-negative MDA-MB-231 cells when co-expressed FOXO3a and ERα, and Ser294Ala-FOXO3a mutant (S294A) is incompetent for TAM-mediated suppression of invasion. Real time qPCR showed that TAM decreased FOXO3a-induced MMP-2 mRNA expression in MCF-7 cells and in MDA-MB-231 cells co-expressed with ERα. Co-immunoprecipitation and immunofluorescent staining showed that Ser294 phosphorylation of FOXO3a might be essential for its nuclear localization and protein-protein interaction with ERα in response to TAM. Conclusion The present study highlights that the real index of nuclear p-ser294-FOXO3a can predict a better survival rate and treatment response of HT and chemotherapy in breast cancer. Furthermore, the specific interplay among p-ser294-FOXO3a, ERα, and MMP-2 might be involved in breast cancer metastasis and treatment response. A better understanding of the underlying mechanism may improve the treatment strategy of specific breast cancer subtype. Citation Format: Ming-Feng Hou, Sheau-Fang Yang, Yu-Chun Huang, Fang-Ming Chen, Yao-Tsung Yeh. The cross-talk between nuclear p-ser294-FOXO3a and cytoplasmic MMP-2 in breast cancer survival and treatment response [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-08-16.

Educated Platelets Promote Wound Healing via Anti-inflammatory Effect and Down-regulated VEGF and MMP-9

Background: This study aims to investigate the effect of educated platelets histological and immunohistochemical (MMP-9 and VEGF) in wound treatment. Methods: 28 female Wistar albino 180-225 gr rats were divided into four groups randomly. The control group (C) is unburnt which blood samples are extracted or PRP with ordinary platelets. Burn group (B) is a burn group in which blood samples were extracted with the educated platelets for PRP. The Burn and uneducated platelet group (B+P) were the burn groups given PRP with uneducated platelets to their blood circulation. Burn and educated platelet group (B+EP) was the burn group given PRP with educated platelets to their blood circulation. The histological analysis was scored 14 days after injury, and MMP-9, VEGF were immunostained. Results: The results clearly showed higher reepithelialization, lower inflammation, and granulation in the B+EP group. Immunohistochemical staining for MMP-9 and VEGF in the B+EP group were statistically lower than other groups. Educated platelet treatment significantly suppressed MMP-9 and VEGF. Conclusion: Our findings suggest that educated-platelet treatment can be a promising strategy for promoting burn cutaneous wound healing. Further and more extensive clinical studies are needed to determine the effectiveness of this treatment method definitively.

Cold exposure and capsaicin promote 1,2-dimethylhyrazine-induced colon carcinogenesis in rats correlates with extracellular matrix remodeling.

BACKGROUNDExtracellular matrix (ECM) remodeling and stiffening, which are correlated with tumor malignancy, drives tumor development. However, the relationship between ECM remodeling and rat experimental model of 1,2-dimethylhyrazine (DMH)-induced colorectal cancer (CRC) imposed by cold and capsaicin exposure remains unclear.AIMTo explore the effects of cold exposure and capsaicin on ECM remodeling and ECM enzymes in DMH-induced CRC.METHODSFor histopathological analysis, the sections of colon tissues were stained with hematoxylin and eosin, Masson’s trichrome, Picrosirius red, and Weigert’s Resorcin-Fuchsin to observe the remodeling of collagen and elastin. Additionally, the protein expression level of type I collagen (COL I), type 3 collagen (COL III0, elastin, matrix metalloproteinase (MMP) 1, MMP2, MMP9, and tissue-specific matrix metalloproteinase 1 (TIMP1) was assessed by immunohistochemistry. The messenger RNA (mRNA) levels of COL I, COL III, elastin, and lysyl oxidase-like-2 (LOXL2) in the colon tissues of rats was measured by reverse-transcriptase quantitative polymerase chain reaction.RESULTSAlthough no differences were observed in the proportion of adenomas, a trend towards the increase of invasive tumors was observed in the cold and capsaicin group. The cold exposure group had a metastasis rate compared with the other groups. Additionally, abnormal accumulation of both collagen and elastin was observed in the cold exposure and capsaicin group. Specifically, collagen quantitative analysis showed increased length, width, angle, and straightness compared with the DMH group. Collagen deposition and straightness were significantly increased in the cold exposure group compared with the capsaicin group. Cold exposure and capsaicin significantly increased the protein levels of COL I, elastin, and LOXL2 along with increases in their mRNA levels in the colon tissues compared with the DMH group, while COL III did not show a significant difference. Furthermore, in immunohistochemical evaluations, MMP1, MMP2, MMP9, and TIMP1 staining increased in the cold exposure and capsaicin group compared with the DMH group.CONCLUSIONThese results suggest that chronic cold and capsaicin exposure further increased the deposition of collagen and elastin in the colonic tissue. Increased COL I and elastin mRNA and protein levels expression may account for the enhanced ECM remodel and stiffness variations of colon tissue. The upregulated expression of the LOXL2 and physiological imbalance between MMP/TIMP activation and deactivation could contribute to the progression of the CRC resulting from cold and capsaicin exposure.

Thrombin and factor Xa promote premature senescence and remodeling in areas at risk of the left appendage and in atrial endothelial cells: potential modulation by SGLT1 and/or SGLT2 inhibition

Abstract Introduction Atrial fibrillation (AF), the most common cardiac arrhythmia, promotes prothrombotic responses particularly in the distal part of the left atrial appendage (LAA) characterized by reduced flow and low shear stress. AF occurrence and duration are closely associated with endothelial dysfunction and left atrium remodeling. The contribution of coagulation factors thrombin and factor Xa (FXa) to LA remodeling remains poorly studied. This study investigated whether thrombin and FXa promote endothelial dysfunction and profibrotic and prothrombotic responses in the distal and proximal parts of the LAA. Since SGLT2 inhibitors have shown pronounced cardiac protection, the contribution of SGLT2 was examined. Methods LAAs were harvested from porcine hearts. Proximal (low stasis, high shear) and distal (high stasis, low shear) parts were cut and incubated with a coagulation factor (thrombin or FXa). Left atrial endothelial cells (AECs) were collected enzymatically, cultured and used at passage 1. The level of oxidative stress was assessed using the redox-sensitive probe dihydroethidium (DHE), fibrosis by Sirius red histological staining, senescence-associated β-galactosidase (SA-β-gal) activity using X-gal staining in tissues and the fluorogenic substrate C12FDG and flow cytometry in cells. The expression of target proteins was detected by immunofluorescence staining and Western blot analysis. Results The distal part of LAA displayed higher levels of oxidative stress in the endothelium and the atrial wall than the proximal part. SA-β-gal activity, ICAM-1, VCAM-1, and MMP-9 staining was more pronounced in the endothelium of the distal part compared to the proximal part whereas eNOS staining was decreased. The distal LAA wall showed higher levels of fibrosis. Western blot analysis showed higher expression levels of tissue factor (TF), VCAM-1, MCP-1, MMP-9, TGF-β and SGLT-2 in the distal part compared to the proximal one. Both thrombin and FXa increased these signals to a greater extent in the distal part of the LAA whereas the eNOS expression level was decreased. Exposure of AECs to either thrombin or FXa induced a pro-oxidant response and increased the expression level of ICAM-1, TGF-β, MMP-2, MMP-9, SGLT1 and SGLT-2. Thrombin promoted SA-β-gal activity in AECs. The selective SGLT-2 inhibitor empagliflozin and the dual SGLT1/2 inhibitor sotagliflozin prevented the pro-oxidant response and SA-β-gal activity in response to thrombin. Conclusion Thus, the distal part of the LAA is prematurely affected by endothelial dysfunction and senescence associated with a pro-oxidant, pro-remodeling and pro-inflammatory response that is further increased by thrombin and FXa. Hence, the coagulation cascade appears to prematurely promote atrial endothelium and wall dysfunction predominantly in areas at risk that may pave the way to thrombus formation. The findings also suggest a potential protective effect of SGLT1 and/or SGLT2 inhibition on atrial dysfunction. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Unrestricted research grant from Boehringer Ingelheim Pharma GmbH &amp; Co. KG, Biberach, Germany

MiR-1293 acts as a tumor promotor in lung adenocarcinoma via targeting phosphoglucomutase 5.

BackgroundLung adenocarcinoma (LUAD) is the most common histologic subtype of lung cancer. Studies have found that miR-1293 is related to the survival of LUAD patients. Unfortunately, its role in LUAD remains not fully clarified.MethodsmiR-1293 expression and its association with LUAD patients’ clinical characteristics were analyzed in TCGA database. Also, miR-1293 expression was detected in LUAD cell lines. Cell viability, migration, invasion and expression of MMP2 and MMP9 were measured in LUAD cells following transfection with miR-1293 mimic or antagomir. Phosphoglucomutase (PGM) 5 was identified to be negatively related to miR-1293 in LUAD patients in TCGA database, and their association was predicated by Targetscan software. Hence, we further verified the relationship between miR-1293 and PGM5. Additionally, the effect and mechanism of miR-1293 were validated in a xenograft mouse model.ResultsWe found miR-1293 expression was elevated, but PGM5 was decreased, in LUAD patients and cell lines. Higher miR-1293 expression was positively related to LUAD patients’ pathologic stage and poor overall survival. miR-1293 mimic significantly promoted, whereas miR-1293 antagomir suppressed the viability, migration, invasion, and expression of MMP2 and MMP9 in LUAD cells. PGM5 was a target of miR-1293. Overexpression of PGM5 abrogated the effects of miR-1293 on the malignant phenotypes of LUAD cells. Administration of miR-1293 antagomir reduced tumor volume and staining of Ki-67 and MMP9, but elevated PGM5 expression in vivo.ConclusionsmiR-1293 promoted the proliferation, migration and invasion of LUAD cells via targeting PGM5, which indicated that miR-1293 might serve as a potential therapeutic target for LUAD patients.

Differences in MMP-2 Expression in High- and Low-Grade MPNST and its Correlation with Prognostic Factors

Malignant peripheral nerve sheath tumor (MPNST) is a soft tissue sarcoma, which is difficult to distinguish from other spindle cell sarcomas. MPNST is hostile, with a high recurrence, and tends to metastasize hematogenously, especially to the lungs. A phase of the metastasis is a degradation of the extracellular matrix, where Matrix Metalloproteinase (MMP) plays an essential role in this process. Gelatinase-type MMP, MMP-2 and MMP-9, can degrade basal membrane and fibrillar collagen to open the invasion pathway. MMP-2 can degrade more collagen and non-collagen extracellular matrix than MMP-9. Therefore, the study aimed to see the relationship between MMP-2 overexpression and histopathological malignancy grading and other clinical prognostic variables. The study was conducted by immunohistochemical staining of MMP-2 in 39 cases, consisting of 19 cases of low-grade MPNST and 20 cases of high-grade MPNST. Subsequently, an analysis of the relationship between MMP-2 overexpression and the malignancy grading and clinical variables was performed, such as age, sex, and tumor size and location. MMP-2 overexpression was seen in 19 (95%) cases of high grade and three (15.8%) cases of low-grade MPNST (p 0.000). The study also found a significant relationship between MMP-2 overexpression and histopathology grading, which may be helpful to define the prognosis.

Tendon and motor phenotypes in the Crtap-/- mouse model of recessive osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is characterized by short stature, skeletal deformities, low bone mass, and motor deficits. A subset of OI patients also present with joint hypermobility; however, the role of tendon dysfunction in OI pathogenesis is largely unknown. Using the Crtap-/- mouse model of severe, recessive OI, we found that mutant Achilles and patellar tendons were thinner and weaker with increased collagen cross-links and reduced collagen fibril size at 1- and 4-months compared to wildtype. Patellar tendons from Crtap-/- mice also had altered numbers of CD146+CD200+ and CD146-CD200+ progenitor-like cells at skeletal maturity. RNA-seq analysis of Achilles and patellar tendons from 1-month Crtap-/- mice revealed dysregulation in matrix and tendon marker gene expression concomitant with predicted alterations in TGF-β, inflammatory, and metabolic signaling. At 4-months, Crtap-/- mice showed increased αSMA, MMP2, and phospho-NFκB staining in the patellar tendon consistent with excess matrix remodeling and tissue inflammation. Finally, a series of behavioral tests showed severe motor impairments and reduced grip strength in 4-month Crtap-/- mice - a phenotype that correlates with the tendon pathology.

Immunohistochemical Expression of MMP-2 in Pancreatic Carcinoma and Chronic Pancreatitis

<i>Introduction:</i> Matrix metalloproteinase (MMP)-2 is a member of MMPs family which is zinc-dependent endopeptidases that degrade all components of ECM and vascular basement membrane. MMPs are closely involved in tumor invasion and metastasis. <i>Aim of the work:</i> is to evaluate the immune-expression and clinical significance of MMPs molecule in patients with pancreatic carcinoma. <i>Materials and Methods:</i> A total of 25 pancreatic carcinomas were evaluated for expression of MMP-2 by immunohistochemical technique. The expression was evaluated. The study included 24/25 cases of PDAC and 1/25 acinar cell carcinoma. PDAC included 5/24 cases of well differentiated, 16/24 cases of moderately differentiated, 3/24 cases of poorly differentiated adenocarcinoma. <i>Results:</i> Pancreatic carcinoma specimens showed positive cytoplasmic MMP-2 staining with high expression in 88% of studied specimens as their IRS ranged from 6/9 to 9/9 with high extent and intensity. There was a statistically significant relationship between MMP-2 expression and high grade in PDAC (p<0.024). <i>Conclusion:</i> MMP-2 was expressed in PDAC and not in pancreatitis cases. The expression of MMP-2 was correlated with high histological grade and poor prognosis in PDAC.

Comparison of the Efficacy and Safety of Trabeculectomy with Mitomycin C According to Concentration: A Prospective Randomized Clinical Trial.

(1) Background: Mitomycin C (MMC) is commonly used during trabeculectomy. However, there is no consensus on which concentration should be used. We aimed to compare the efficacy and safety of 0.2 mg/mL and 0.4 mg/mL of MMC in eyes undergoing trabeculectomy. (2) Methods: Thirty-six eyes (36 glaucoma patients) were randomized to undergo a trabeculectomy with 0.2 mg/mL or 0.4 mg/mL of MMC. The success rate was evaluated according to three criteria: (A) intraocular pressure (IOP) ≤ 18 mmHg and IOP reduction ≥ 20%; (B) IOP ≤ 15 mmHg and IOP reduction ≥ 25%; (C) IOP ≤ 12 mmHg and IOP reduction ≥ 30%. Cox’s proportional hazard model was used to identify the predictive factors for failure. Immunohistochemical procedures for matrix metalloproteinase (MMP) were performed on Tenon’s tissue. Bleb morphology was evaluated. Safety was assessed based on the incidence of complications. (3) Results: Of the 36 eyes, 19 underwent trabeculectomy with 0.2 mg/mL of MMC and 17 with 0.4 mg/mL. The success rates were 75%, 67%, and 47% at 6 months for criteria A, B, and C, respectively. There were no significant differences between the two groups. High MMP-9 staining and low preoperative IOP were associated with failure (hazard ratio (HR), 5.556; p = 0.033, and HR, 0.936; p = 0.033). Complications included hypotony in two eyes (6%), hyphema in one eye (3%), and choroidal detachment in one eye (3%). (4) Conclusions: Trabeculectomy with 0.2 mg/mL and 0.4 mg/mL of MMC showed similar IOP-control effects to those recorded in previous studies, along with a low rate of complications. There was no significant difference in efficacy or safety between the 0.2 mg/mL and 0.4 mg/mL MMC groups.

TExpression of matrix metalloproteinases 1, 2, 9, 12 in xenogenic tissues of epoxy-crosslinked bioprosthetic heart valves explanted due to dysfunction

Aim. To study the expression patterns of matrix metalloproteinases (MMPs) -1, -2, -9, -12 in the leaflets of the epoxy-treated bioprostheses explanted due to dysfunction and to identify the pathways for the accumulation of these enzymes in the xenogenic tissues.Methods. 19 leaflets from seven epoxy-treated bioprostheses (Kemcor (n = 2), PeriCor (n = 2), UniLine (n = 2) and TiAra (n = 1)) explanted from the mitral or aortic positions during repeat heart valve replacements were included in a study. Sections for microscopic studies were cut on a standard rotary microtome. Cell typing and the expression of MMP-1, -2, -9, -12 were evaluated using immunohistochemical staining with the antibodies against PTPRC/CD45, CD68, neutrophil myeloperoxidase and the corresponding MMPs. Stained samples were examined by light microscopy.Results. Sporadic cell infiltrates, mainly composed of macrophages (PTPRC/CD45+, CD68+), were found in 17 leaflets from six explanted bioprostheses. Positive staining for MMP-1, -2, -9, -12 was colocalized with immune cell infiltrates. It is worth noting that MMP-9 staining was visualized even in the absence of cell infiltration, while more intense staining was found in the areas with a loose extracellular matrix. There were no signs of macrophage infiltration or MMP expression in xenotissues of pericardial bioprostheses failed due to thrombosis and explanted two days after implantation. However, a blood clot formed on its surface showed intense MMP-9 staining and included a large proportion of neutrophils positive for myeloperoxidase.Conclusion. Macrophages and other immune cells that infiltrate xenotissues of epoxy-treated bioprostheses are sources of MMP-1, -2, -9, -12. In addition, MMP-9 can diffuse into bioprosthetic valve leaflets from blood plasma of patients. Thus, MMPs deposition in xenotissues may contribute to the leaflet ruptures and calcifications leading to the development of bioprosthetic valve dysfunction.