Neointimal hyperplasia after endoluminal injury in mice is dependent on tissue factor- and angiopoietin-2 dependent interferon gamma production by fibrocytes and macrophages.

Background: The intimal hyperplasia (IH) and vascular remodelling that follows endovascular injury, for instance after post-angioplasty re-stenosis, results in downstream ischaemia and progressive end organ damage. Interferon gamma (IFNγ) is known to play a critical role in this process. In mouse models we have previously shown that fibrocytes expressing tissue factor (TF) are recruited early to the site of injury. Through thrombin generation and protease activated receptor-1 (PAR-1) activation, fibrocytes secrete angiopoietin-2, stimulate neointimal cell proliferation, inhibit apoptosis and induce CXCL-12 production, all of which contribute to the progressive IH that then develops. In this study we investigated the relationship between TF, angiopoietin-2 and IFNγ. Methods and results: IH developing in carotid arteries of wild-type mice 4 weeks after endoluminal injury contained a significant proportion of IFNγ+ fibrocytes and macrophages, which we show, using a previously defined adoptive transfer model, were derived from circulating CD34+ cells. IH did not develop after injury in IFNγ-deficient mice, except after transplantation of WT bone marrow or adoptive transfer of WT CD34+ cells. In vitro, CD34+ cells isolated from post-injury mice did not express IFNγ, but this was induced when provided with FVIIa and FX, and enhanced when prothrombin was also provided: In both cases IFNγ secretion was TF-dependent and mediated mainly through protease activated PAR-1. IFNγ was predominantly expressed by fibrocytes. In vivo, all IFNγ+ neointimal cells in WT mice co-expressed angiopoietin-2, as did the small numbers of neointimal cells recruited in IFNγ-/- mice. Adoptively transferred WT CD34+ cells treated with either an anti-TIE-2 antibody, or with siRNA against angiopoetin-2 inhibited the expression of IFNγ and the development of IH. Conclusion: TF-dependent angiopoietin-2 production by newly recruited fibrocytes, and to a lesser extent macrophages, switches on IFNγ expression, and this is necessary for the IH to develop. These novel findings enhance our understanding of the pathophysiology of IH and expose potential targets for therapeutic intervention.

Qingda granule alleviates cerebral ischemia/reperfusion injury by inhibiting TLR4/NF-κB/NLRP3 signaling in microglia.

ETHNOPHARMACOLOGICAL RELEVANCE: Qingda granule (QDG) is effective for treating hypertension and neuronal damage after cerebral ischemia/reperfusion. However, the anti-neuroinflammatory effect of QDG on injury due to cerebral ischemia/reperfusion is unclear. AIM OF THE STUDY: The objective was to evaluate the effectiveness and action of QDG in treating neuroinflammation resulting from cerebral ischemia/reperfusion-induced injury. MATERIALS AND METHODS: Network pharmacology was used to predict targets and pathways of QDG. An in vivo rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) as well as an in vitro model of LPS-stimulated BV-2 cells were established. Magnetic resonance imaging (MRI) was used to quantify the area of cerebral infarction, with morphological changes in the brain being assessed by histology. Immunohistochemistry (IHC) was used to assess levels of the microglial marker IBA-1 in brain tissue. Bioplex analysis was used to measure TNF-α, IL-1ß, IL-6, and MCP-1 in sera and in BV-2 cell culture supernatants. Simultaneously, mRNA levels of these factors were examined using RT-qPCR analysis. Proteins of the TLR4/NF-κB/NLRP3 axis were examined using IHC in vivo and Western blot in vitro, respectively. While NF-κB translocation was assessed using immunofluorescence. RESULTS: The core targets of QDG included TNF, NF-κB1, MAPK1, MAPK3, JUN, and TLR4. QDG suppressed inflammation via modulation of TLR4/NF-κB signaling. In addition, our in vivo experiments using MCAO/R rats demonstrated the therapeutic effect of QDG in reducing brain tissue infarction, improving neurological function, and ameliorating cerebral histopathological damage. Furthermore, QDG reduced the levels of TNF-α, IL-1ß, IL-6, and MCP-1 in both sera from MCAO/R rats and supernatants from LPS-induced BV-2 cells, along with a reduction in the expression of the microglia biomarker IBA-1, as well as that of TLR4, MyD88, p-IKK, p-IκBα, p-P65, and NLRP3 in MCAO/R rats. In LPS-treated BV-2 cells, QDG downregulated the expression of proinflammatory factors and TLR4/NF-κB/NLRP3 signaling-related proteins. Additionally, QDG reduced translocation of NF-κB to the nucleus in both brains of MCAO/R rats and LPS-induced BV-2 cells. Moreover, the combined treatment of the TLR4 inhibitor TAK242 and QDG significantly reduced the levels of p-P65, NLRP3, and IL-6. CONCLUSIONS: QDG significantly suppressed neuroinflammation by inhibiting the TLR4/NF-κB/NLRP3 axis in microglia. This suggests potential for QDG in treating ischemia stroke.

Liensinine improves AngII-induced vascular remodeling via MAPK/TGF-ß1/Smad2/3 signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Liensinine(Lien, C37H42N2O6) is an alkaloid compound from plumula nelumbinis that demonstrates an antihypertensive effect. The protective effects of Lien on target organs during hypertension are still unclear. AIM OF THE STUDY: This study aimed to understand the mechanism of Lien during the treatment of hypertension, with emphasis on vascular protection. MATERIALS AND METHODS: Lien was extracted and isolated from plumula nelumbinis for further study. In vivo model of Ang II-induced hypertension, non-invasive sphygmomanometer was used to detect the blood pressure in and out of the context of Lien intervention. Ultrasound was used to detect the abdominal aorta pulse wave and media thickness of hypertensive mice, and RNA sequencing was used to detect the differential genes and pathways of blood vessels. The intersection of Lien and MAPK protein molecules was detected by molecular interconnecting technique. The pathological conditions of abdominal aorta vessels of mice were observed by HE staining. The expression of PCNA, α-SMA, Collagen Type Ⅰ and Collagen Type Ⅲ proteins were detected by IHC. The collagen expression in the abdominal aorta was detected by Sirius red staining. The MAPK/TGF-ß1/Smad2/3 signaling and the protein expression of PCNA and α-SMA was detected by Western blot. In vitro, MAPK/TGF-ß1/Smad2/3 signaling and the protein expression of PCNA and α-SMA were detected by Western blot, and the expression of α-SMA was detected by immunofluorescence; ELISA was used to detect the effect of ERK/MAPK inhibitor PD98059 on Ang Ⅱ-induced TGF-ß1secrete; and the detection TGF-ß1and α-SMA protein expression by Western blot; Western blot was used to detect the effect of ERK/MAPK stimulant12-O-tetradecanoyl phorbol-13-acetate (TPA) on the protein expression of TGF-ß1 and α-SMA. RESULTS: Lien displayed an antihypertensive effect on Ang Ⅱ-induced hypertension, reducing the pulse wave conduction velocity of the abdominal aorta and the thickness of the abdominal aorta vessel wall, ultimately improving the pathological state of blood vessels. RNA sequencing further indicated that the differential pathways expressed in the abdominal aorta of hypertensive mice were enriched in proliferation-related markers compared with the Control group. The profile of differentially expressed pathways was ultimately reversed by Lien. Particularly, MAPK protein demonstrated good binding with the Lien molecule. In vivo, Lien inhibited Ang Ⅱ-induced abdominal aorta wall thickening, reduced collagen deposition in the ventral aortic vessel, and prevented the occurrence of vascular remodeling by inhibiting MAPK/TGF-ß1/Smad2/3 signaling activation. In addition, Lien inhibited the activation of Ang II-induced MAPK and TGF-ß1/Smad2/3 signaling, attenuating the expression of PCNA and inhibiting the reduction of α-SMA, collectively playing a role in the inhibition of Ang Ⅱ-induced hypertensive vascular remodeling. PD98059 alone could inhibit Ang Ⅱ-induced elevation of TGF-ß1 and the decrease of α-SMA expression. Further, PD98059 combined with Lien had no discrepancy with the inhibitors alone. Simultaneously TPA alone could significantly increase the expression of TGF-ß1 and decrease the expression of α-SMA. Further, Lien could inhibit the effect of TPA. CONCLUSION: This study helped clarify the protective mechanism of Lien during hypertension, elucidating its role as an inhibitor of vascular remodeling and providing an experimental basis for the research and development of novel antihypertensive therapies.

Qingda granule alleviate angiotensin â ±-induced hypertensive renal injury by suppressing oxidative stress and inflammation through NOX1 and NF-κB pathways.

Hypertension has become one of the important diseases harmful to human health. In China, Qingda granule (QDG) has been used to treat hypertension for decades. Previous studies by our team have shown that oxidative stress may be one of the pathways through which QDG inhibits hypertension-induced organs injury. However, the specific molecular mechanism of its anti-hypotension and renal oxidative stress response were unclearly. This study investigated QDG's potential protective mechanism against hypertension-induced renal injury. Mice were infused with Angiotensin Ⅱ (Ang Ⅱ, 500 ng/kg/min) or equivalent saline solution (Control) and administered oral QDG (1.145 g/kg/day) or saline for four weeks. QDG treatment mitigated the elevated blood pressure and reduced renal pathological changes induced by Ang Ⅱ. As per the RNA sequencing results, QDG affects oxidative stress signaling. In agreement with these findings, QDG significantly attenuated the Ang Ⅱ-induced increase in Nitrogen oxides 1 (NOX1) and reactive oxygen species and the decrease in superoxide dismutase in renal tissue. Additionally, QDG significantly inhibited Interleukin 6 (IL-6), Tumor necrosis factor α (TNF-α), and Interleukin 1ß (IL-1ß) expression in renal tissues and blocked the phosphorylation of P65 (NF-κB subunit) and IκB. These results were confirmed in vitro. Overall, QDG reduced Ang Ⅱ-induced elevated blood pressure and renal injury by inhibiting oxidative stress and inflammation caused by NOX1 and NF-κB pathways. The results of this study provide an experimental basis for the clinical application of QDG, and to open up a new direction for the clinical treatment of hypertension.

Manipulation of tissue factor-mediated basal PAR-2 signalling on macrophages determines sensitivity for IFNγ responsiveness and significantly modifies the phenotype of murine DTH.

Background: Tissue factor (TF) generates proteases that can signal through PAR-1 and PAR-2. We have previously demonstrated PAR-1 signalling primes innate myeloid cells to be exquisitely sensitive to interferon-gamma (IFNγ). In this work we explored how TF mediated PAR-2 signalling modulated responsiveness to IFNγ and investigated the interplay between PAR-1/-2 signalling on macrophages. Methodology: We characterised how TF through PAR-2 influenced IFNγ sensitivity in vitro using PCR and flow cytometry. and how it influenced oxazolone-induced delayed type hypersensitivity (DTH) responses in vivo. We investigated how basal signalling through PAR-2 influenced PAR-1 signalling using a combination of TF-inhibitors and PAR-1 &-2 agonists and antagonists. Finally, we investigated whether this system could be targeted therapeutically using 3-mercaptopropionyl-F-Cha-Cha-RKPNDK (3-MP), which has actions on both PAR-1 and -2. Results: TF delivered a basal signal through PAR-2 that upregulated SOCS3 expression and blunted M1 polarisation after IFNγ stimulation, opposing the priming achieved by signalling through PAR-1. PAR-1 and -2 agonists or antagonists could be used in combination to modify this basal signal in vitro and in vivo. 3-MP, by virtue of its PAR-2 agonist properties was superior to agents with only PAR-1 antagonist properties at reducing M1 polarisation induced by IFNγ and suppressing DTH. Tethering a myristoyl electrostatic switch almost completely abolished the DTH response. Conclusions: TF-mediated signalling through PARs-1 and -2 act in a homeostatic way to determine how myeloid cells respond to IFNγ. 3-MP, an agent that simultaneously inhibits PAR-1 whilst delivering a PAR-2 signal, can almost completely abolish immune responses dependent on M1 polarisation, particularly if potency is enhanced by targeting to cell membranes; this has potential therapeutic potential in multiple diseases.

LINC01006 and miR-3199 Serve as Novel Markers of Poor Prognosis in Colon Cancer and Regulate Cell Proliferation, Migration and Invasion.

PURPOSE: Colon cancer is the most commonly diagnosed gastrointestinal cancer. This research intended to evaluate the prognostic values of LINC01006 and miR-3199 for colon cancer and their effects on cell physiology. PATIENTS AND METHODS: LINC01006 and miR-3199 expression levels were determined by RT-qPCR. Patients' 5-year cumulative survival rate was analyzed by Kaplan-Meier curves with the Log rank test. Chi-square test and multivariate Cox regression analysis were used to access the clinical significance. CCK-8 assay, transwell assay, and TUNEL assays were used to monitor the change of cell proliferation, invasion, migration, and apoptosis. RESULTS: The expression level of LINC01006 was increased while miR-3199 was decreased in colon tissues and cells compared to normal ones. This dysregulated expression was correlated with T stage (P = 0.002) and N stage (P = 0.009). High LINC01006 level (HR = 4.048, 95%: 1.502-10.911, P = 0.006) or low miR-3199 level (HR = 3.421, 95% CI: 1.254-9.330, P = 0.016) was outstanding for predicting poor prognosis in patients with colon cancer. Downregulation of LINC01006 reduced cell proliferation, invasion, and migration but induced cell apoptosis (P < 0.05). CONCLUSION: LINC01006 knockdown showed anti-proliferative, anti-metastatic, and apoptotic-induced effects on colon cancer cells. This study contributes to research on promising prognostic biomarkers of colon cancer and might give way to further investigation of alternative tumor targets.

Therapeutic Effects of Different Animal Bile Powders on Lipid Metabolism Disorders and Their Composition Analysis.

OBJECTIVE: To compare the therapeutic effect of different animal bile powders on lipid metabolism disorders induced by high-fat diet in rats, and analyze the bioactive components of each animal bile powder. METHODS: Sixty Sprague-Dawley rats were randomly divided into 6 groups (n=10): normal diet control group, high-fat diet model group, high-fat diet groups orally treated with bear, pig, cow and chicken bile powders, respectively. Serum biochemical markers from the abdominal aorta in each group were analyzed. Changes in the body weight and liver weight were recorded. Pathohistological changes in the livers were examined. High performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry was used to determine the composition of bioactive components in each animal bile powder. RESULTS: Treatment with different types of animal bile powders had different inhibitory effects on high-fat diet-induced increase of body weight and/or liver weight in rats, most notably in bear and pig bile powders (P<0.05). High-fat diet induced lipid metabolism disorder in rats, which could be reversed by treatment with all kinds of bile powders. Bear bile and chicken bile showed the most potent therapeutic effect against lipid metabolism disorder. Cow and bear bile effectively alleviated high-fat diet induced liver enlargement and discoloration, hepatocyte swelling, infiltration of inflammatory cells and formation of lipid vacuoles. Bioactive component analysis revealed that there were significant differences in the relative content of taurocholic acid, taurodeoxycholic acid and ursodeoxycholic acid among different types of animal bile. Interestingly, a unique component with molecular weight of 496.2738 Da, whose function has not yet been reported, was identified only in bear bile powder. CONCLUSIONS: Different animal bile powders had varying therapeutic effect against lipid metabolism disorders induced by high-fat diet, and bear bile powder demonstrated the most effective benefits. Bioactive compositions were different in different types of animal bile with a novel compound identified only in bear bile powder.

Cardiac biomarkers, cardiac injury, and comorbidities associated with severe illness and mortality in coronavirus disease 2019 (COVID-19): A systematic review and meta-analysis.

AIMS: To explore the correlation between cardiac-related comorbidities, cardiac biomarkers, acute myocardial injury, and severity level, outcomes in COVID-19 patients. METHOD: Pubmed, Web of Science, Embase, CNKI, VIP, Wanfang, Cochrane Library databases, medRxiv, and Sinomed were reviewed systemically. Various types of clinical research reporting cardiac-related comorbidities, cardiac biomarkers including lactate dehydrogenase (LDH), troponin I (TnI), high sensitivity troponin I (hs-TnI), creatine kinase (CK), creatine kinase-MB (CK-MB), myoglobin (Myo), N-terminal pro-b-type natriuretic peptide (NT-proBNP) and acute cardiac injury grouped by severity of COVID-19 were included. Outcome measures were events and total sample size for comorbidities, acute cardiac injury, and laboratory parameters of these biomarkers. The study was performed with Stata version 15.1. RESULTS: Seventy studies, with a total of 15,354 cases were identified. The results showed that COVID-19's severity was related to cardiovascular disease. Similar odds ratios (ORs) were achieved in hypertension except for severe versus critical group (OR = 1.406; 95% CI, 0.942-2.097; p = .095). The relative risk (RR) of acute cardiac injury is 7.01 (95% CI, 5.64-8.71) in non-survivor cases. When compared with the different severity of cardiac biomarkers, the pool OR of CK, CK-MB, TnI, Myo and LDH were 2.683 (95% CI, 0.83-8.671; p = .106; I2 = 0%), 2.263 (95% CI, 0.939-5.457; p = .069), 1.242 (95% CI, 0.628-2.457; p = .534), 1.756 (95% CI, 0.608-5.071; p = .298; I2 = 42.3%), 1.387 (95% CI, 0.707-2.721;  p = .341; I2 = 0%) in the critical versus severe group, whose trends were not similar to other groups. The standard mean differences (SMD) of CK and TnI in the critical versus severe group were 0.09 (95% CI, -0.33 to 0.50; p = .685; I2 = 65.2%), 0.478 (95% CI, -0.183 to 1.138; p = .156; I2 = 76.7%), which means no difference was observed in the serum level of these indicators. CONCLUSION: Most of the findings clearly indicate that hypertension, cardiovascular disease, acute cardiac injury, and related laboratory indicators are associated with the severity of COVID-19. What is now needed are cross-national prospectively designed observational or clinical trials that will help improve the certainty of the available evidence and treatment decisions for patients.

Comprehensive chemical study on different organs of cultivated and wild Sarcandra glabra using ultra-high performance liquid chromatography time-of-flight mass spectrometry (UHPLC-TOF-MS).

To illuminate the similarities and differences between wild and cultivated Sarcandra glabra (S. glabra), we performed a comprehensively study on 26 batches of cultivated S. glabra and 2 batches of wild S. glabra. Chemical constituents and distribution characteristics of roots, stems and leaves in both wild and cultivated S. glabra were investigated through UHPLC-TOF-MS method. The result revealed that there were significant differences between roots, stems and leaves in S. glabra. And the chemical contents in the root part were less or even absence than those in leaf and stem, which suggested the root organ could be excluded as medicine. Meanwhile, the chemical contents of stems and leaves in cultivated S. glabra was sightly higher than that of wild samples. Therefore, cultivated S. glabra may have a high potential for substitution of wild S. glabra without affecting its pharmaceutical properties. In summary, our study could provide important information to the molecular basis for quality control of S. glabra.

Huoxin Pill inhibits isoproterenol-induced transdifferentiation and collagen synthesis in cardiac fibroblasts through the TGF-ß/Smads pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: The abnormal proliferation and differentiation of cardiac fibroblasts (CFs) are universally regarded as the key process for the progressive development of cardiac fibrosis following various cardiovascular diseases. Huoxin Pill (Concentrated pill, HXP) is a Chinese herbal formula for treating coronary heart disease. However, the cellular and molecular mechanisms of HXP in the treatment of myocardial fibrosis are still unclear. AIM OF THE STUDY: To investigate the effects of HXP on CFs transdifferentiation and collagen synthesis under isoproterenol (ISO) conditions, as well as the potential mechanism of action. MATERIALS AND METHODS: In vivo, we established a rat model of cardiac fibrosis induced by ISO, and administered with low or high dose of HXP (10 mg/kg/day or 30 mg/kg/day). The level of α-SMA was detected by immunohistochemistry examination, and combined with RNA-sequencing analysis to determine the protective effect of HXP on myocardial fibrosis rats. In vitro, by culturing primary rat CFs, we examined the effects of HXP on the proliferation and transdifferentiation of CFs using CCK8, scratch wound healing and immunofluorescence assays. Western blot was used to determine protein expression. RESULTS: The findings revealed that HXP protects against ISO-induced cardiac fibrosis and CFs transdifferentiation in rats. RNA-sequencing and pathway analyses demonstrated 238 or 295 differentially expressed genes (DEGs) and multiple enriched signal pathways, including transforming growth factor-beta (TGF-ß) receptor signaling activates Smads, downregulation of TGF-ß receptor signaling, signaling by TGF-ß receptor complex, and collagen formation under treatment with low or high-dose of HXP. Moreover, HXP also markedly inhibited ISO-induced primary rat CFs proliferation, transdifferentiation, collagen synthesis and the upregulation of TGF-ß1 and phosphorylated Smad2/3 protein expression. CONCLUSION: HXP suppresses ISO-induced CFs transdifferentiation and collagen synthesis, and it may exert these effects in part by inhibiting the activation of the TGF-ß/Smads pathway. This may be a new therapeutic tool for cardiac fibrosis.

PAR-1 signaling on macrophages is required for effective in vivo delayed-type hypersensitivity responses.

Delayed-type hypersensitivity (DTH) responses underpin chronic inflammation. Using a model of oxazolone-induced dermatitis and a combination of transgenic mice, adoptive cell transfer, and selective agonists/antagonists against protease activated receptors, we show that that PAR-1 signaling on macrophages by thrombin is required for effective granuloma formation. Using BM-derived macrophages (BMMs) in vitro, we show that thrombin signaling induced (a) downregulation of cell membrane reverse cholesterol transporter ABCA1 and (b) increased expression of IFNγ receptor and enhanced co-localization within increased areas of cholesterol-rich membrane microdomains. These two key phenotypic changes combined to make thrombin-primed BMMs sensitive to M1 polarization by 1000-fold less IFNγ, compared to resting BMMs. We confirm that changes in ABCA1 expression were directly responsible for the exquisite sensitivity to IFNγ in vitro and for the impact on granuloma formation in vivo. These data indicate that PAR-1 signaling plays a hitherto unrecognized and critical role in DTH responses.

Protective Role of C3aR (C3a Anaphylatoxin Receptor) Against Atherosclerosis in Atherosclerosis-Prone Mice.

OBJECTIVE: Emerging evidence suggests that C3aR (C3a anaphylatoxin receptor) signaling has protective roles in various inflammatory-related diseases. However, its role in atherosclerosis has been unknown. The purpose of the study was to investigate the possible protective role of C3aR in aortic atherosclerosis and explore molecular and cellular mechanisms involved in the protection. Approach and Results: C3ar-/-/Apoe-/- mice were generated by cross-breeding of atherosclerosis-prone Apoe-/- mice and C3ar-/- mice. C3ar-/-/Apoe-/- mice and Apoe-/- mice (as a control) underwent high-fat diet for 16 weeks were assessed for (1) atherosclerotic plaque burden, (2) aortic tissue inflammation, (3) recruitment of CD11b+ leukocytes into atherosclerotic lesions, and (4) systemic inflammatory responses. Compared with Apoe-/- mice, C3ar-/-/Apoe-/- mice developed more severe atherosclerosis. In addition, C3ar-/-/Apoe-/- mice have increased local production of proinflammatory mediators (eg, CCL2 [chemokine (C-C motif) ligand 2], TNF [tumor necrosis factor]-α) and infiltration of monocyte/macrophage in aortic tissue, and their lesional macrophages displayed an M1-like phenotype. Local pathological changes were associated with enhanced systemic inflammatory responses (ie, elevated plasma levels of CCL2 and TNF-α, increased circulating inflammatory cells). In vitro analyses using peritoneal macrophages showed that C3a stimulation resulted in upregulation of M2-associated signaling and molecules, but suppression of M1-associated signaling and molecules, supporting the roles of C3a/C3aR axis in mediating anti-inflammatory response and promoting M2 macrophage polarization. CONCLUSIONS: Our findings demonstrate a protective role for C3aR in the development of atherosclerosis and suggest that C3aR confers the protection through C3a/C3aR axis-mediated negative regulation of proinflammatory responses and modulation of macrophage toward the anti-inflammatory phenotype.

Regression of Atherosclerosis in ApoE-/- Mice Via Modulation of Monocyte Recruitment and Phenotype, Induced by Weekly Dosing of a Novel "Cytotopic" Anti-Thrombin Without Prolonged Anticoagulation.

Background Anticoagulants induce atherosclerosis regression in animal models but exploiting this clinically is limited by bleeding events. Here we test a novel thrombin inhibitor, PTL060, comprising hirulog covalently linked to a synthetic myristoyl electrostatic switch to tether to cell membranes. Methods and Results ApoE-/- mice were fed chow or high-fat diets, before transplantation of congenic aortic segments or injection of PTL060, parental hirulog, control saline, or labeled CD11b positive cells. Aortic transplants from transgenic mice expressing anticoagulants on endothelium did not develop atherosclerosis. A single intravenous injection of PTL060, but not hirulog inhibited atheroma development by >50% compared with controls when assessed 4 weeks later. Mice had prolonged bleeding times for only one seventh of the time that PTL060 was biologically active. Repeated weekly injections of PTL060 but not hirulog caused regression of atheroma. We dissected 2 contributory mechanisms. First, the majority of CCR2+ (C-C chemokine receptor type 2+) monocytes recruited into plaques expressed CCR7 (C-C chemokine receptor type 7), ABCA1 (ATP-binding cassette transporter - 1), and interleukin-10 in PTL060 mice, a phenotype seen in <20% of CCR2+ recruits in controls. Second, after several doses, there was a significant reduction in monocyte recruits, the majority of which were CCR2-negative with a similar regression-associated phenotype. Regression equivalent to that induced by intravenous PTL060 was induced by adoptive transfer of CD11b+ cells pre-coated with PTL060. Conclusions Covalent linkage of a myristoyl electrostatic switch onto hirulog in PTL060 uncouples the pharmacodynamic effects on hemostasis and atherosclerosis, such that plaque regression, mediated predominantly via effects on monocytes, is accompanied by only transient anticoagulation.

Comparison of long-term outcomes of laparoscopic and open sphincter-preserving total mesorectal excision for low rectal cancer.

PURPOSE: The purpose of this study was to compare the long-term outcomes of laparoscopic and open sphincter-preserving total mesorectal excision (TME) for low rectal cancer (LRC) using propensity score matching (PSM). METHODS: The clinical and follow-up data of 169 patients with LRC who underwent sphincter-preserving TME at our institution between January 2011 and January 2014 were retrospectively analyzed. Patients were divided into laparoscopic and open group based on the surgical approach. PSM including age, sex, body mass index, clinical stage, and American Society of Anesthesiologists score with a 1:1 ratio was subsequently performed. Sixty-eight patients in each group were ultimately included, and short- and long-term outcomes were compared between groups. RESULTS: Compared with the open group, the laparoscopic group had less intraoperative blood loss, more rapid postoperative recovery, and lower incidence of 30-day postoperative complications. However, there were no significant differences in severity of postoperative 30-day complications between the two groups. Both groups had no intraoperative or 30-day postoperative mortality. Regarding survival outcome, tumor recurrence rate, tumor recurrence site, 5-year overall survival, and 5-year disease-free survival, there were no significant differences between groups. CONCLUSION: Laparoscopic sphincter-preserving TME can achieve long-term outcomes similar to those of open TME for LRC.

Qingda granules attenuate hypertensive cardiac remodeling and inflammation in spontaneously hypertensive rats.

Qingda granules (QDG) are derived from QingXuanJiangYa Decoction (QXJYD) a traditional Chinese medication that has been used to treat hypertension for more than 60 years. QXJYD has been shown to be effective in rat models of hypertension. However, the effects of QDG on hypertension remain largely unknown. In the current study, baicalin was identified as one of the main components of QDG using Ultra Performance Liquid Chromatography (UPLC) analysis. We investigated the effects of QDG on blood pressure, cardiac remodeling, and cardiac inflammation. QDG (0.8 g/kg/day) treatment attenuated the elevated blood pressure in spontaneously hypertensive rats (SHRs). Moreover, QDG treatment reduced the degree of myocardial fiber disarray, degeneration and necrosis of myocardial cells, expression of ANP and BNP, as well as collagen content of SHRs. Moreover, we further assessed the effect of QDG treatment on cardiac inflammation and found that QDG treatment reduced CD68 protein expression, decreased levels of IL-6 and TNF-α in both serum and cardiac tissues, as well as suppressed activation of NF-κB pathway in cardiac tissues of SHRs. Differential expressed metabolites (DEMs) analysis identified 41 increased and 51 decreased metabolites in the cardiac tissues of SHRs after QDG treatment. In summary, QDG treatment of SHRs attenuated the elevated blood pressure and ameliorated cardiac remodeling and inflammation, in part, through suppression of NF-κB pathway and DEMs, which provide a basis for other therapeutic uses of this TCM.

LRP6 Ectodomain Prevents SDF-1/CXCR4-Induced Breast Cancer Metastasis to Lung.

PURPOSE: Lung metastasis is an important cause of breast cancer-related deaths, in which SDF-1/CXCR4 signaling pathway plays a critical role. Single transmembrane protein LRP6 is viewed as an oncogene via activating the Wnt/ß-catenin signaling pathway. Our work aims to investigate the relationship between SDF-1/CXCR4 and LRP6 in breast cancer lung metastasis. EXPERIMENTAL DESIGN: We examined the expressions and functions of SDF-1/CXCR4 and LRP6 as well as their relationship in breast cancer in vitro and in vivo. RESULTS: LRP6 ectodomain (LRP6N) directly bound to CXCR4 and competitively prevented SDF-1 binding to CXCR4. LRP6N prevented SDF-1/CXCR4-induced metastasis to lung and prolonged survival in mice bearing breast tumors, whereas LRP6 knockdown activated SDF-1/CXCR4 signal transduction and promoted lung metastasis and tumor death. Furthermore, patients with breast cancer with high CXCR4 expression had poor prognosis, which was exacerbated by low LRP6 expression but improved by high LRP6 expression. Interestingly, a secreted LRP6N was found in the serum of mice and humans, which was downregulated by the onset of cancer metastasis in both mice bearing breast cancer as well as in patients with breast cancer. CONCLUSIONS: LRP6N might be a promising diagnostic marker for the early detection of breast cancer metastasis as well as an inhibitor of SDF-1/CXCR4-induced breast cancer metastasis. LRP6N also provides an interesting link between Wnt signaling and SDF-1/CXCR4 signaling, the two key pathways involved in cancer development.

Shexiang Tongxin Dropping Pill () Protects against Na2S2O4-Induced Hypoxia-Reoxygenation Injury in H9c2 Cells.

OBJECTIVES: To investigate the protective effects of Shexiang Tongxin Dropping Pill (, STP) on Na2S2O4-induced hypoxia-reoxygenation injury in cardiomyoblast H9c2 cells. METHODS: The cell viability and levels of mRNA and protein expression in H9c2 cells were determined following Na2S2O4-induced hypoxia using Hoechst staining, annexin V/propidium iodide (PI) flow cytometry, real-time polymerase chain reaction and Western blot analysis. RESULTS: STP pretreatment significantly increased the viability and inhibited aberrant morphological changes in H9c2 cardiomyoblast cells induced by Na2S2O4 treatment (P<0.05). In addition, STP pretreatment attenuated Na2S2O4-induced hypoxic damage, down-regulated the expression of pro-apoptotic Bax, and up-regulated the expression of anti-apoptotic Bcl-2 in H9c2 cells (P<0.05). CONCLUSIONS: STP was strongly cardioprotective in hypoxia-reoxygenation injury by preventing hypoxic damage and inhibiting cellular apoptosis. These results further support the use of STP as an effective drug for the treatment of ischemic heart disease.

Endothelial cell-specific anticoagulation reduces inflammation in a mouse model of acute lung injury.

Tissue factor (TF)-dependent coagulation contributes to lung inflammation and the pathogenesis of acute lung injury (ALI). In this study, we explored the roles of targeted endothelial anticoagulation in ALI using two strains of transgenic mice expressing either a membrane-tethered human tissue factor pathway inhibitor (hTFPI) or hirudin fusion protein on CD31+ cells, including vascular endothelial cells (ECs). ALI was induced by intratracheal injection of LPS, and after 24 h the expression of TF and protease-activated receptors (PARs) on EC in lungs were assessed, alongside the extent of inflammation and injury. The expression of TF and PARs on the EC in lungs was upregulated after ALI. In the two strains of transgenic mice, expression of either of hTFPI or hirudin by EC was associated with significant reduction of inflammation, as assessed by the extent of leukocyte infiltration or the levels of proinflammatory cytokines, and promoted survival after LPS-induced ALI. The beneficial outcomes were associated with inhibition of the expression of chemokine CCL2 in lung tissues. The protection observed in the CD31-TFPI-transgenic strain was abolished by injection of an anti-hTFPI antibody, but not by prior engraftment of the transgenic strains with WT bone marrow, confirming that the changes observed were a specific transgenic expression of anticoagulants by EC. These results demonstrate that the inflammation in ALI is TF and thrombin dependent, and that expression of anticoagulants by EC significantly inhibits the development of ALI via repression of leukocyte infiltration, most likely via inhibition of chemokine gradients. These data enhance our understanding of the pathology of ALI and suggest a novel therapeutic strategy for treatment.

Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice.

Fibrocytes are myeloid lineage cells implicated in wound healing, repair, and fibrosis. We previously showed that fibrocytes are mobilized into the circulation after vascular injury, including the immune-mediated injury that occurs after allogeneic transplantation. A common response to inflammatory vascular injury is intimal hyperplasia (IH), which, alongside vascular remodeling, results in progressive loss of blood flow, downstream ischemia, and end-organ fibrosis. This forms the pathological basis of transplant arteriosclerosis and other diseases including post-angioplasty re-stenosis. In investigating whether fibrocytes contribute to IH, we previously showed that subpopulations expressing smooth muscle actin and CD31 are recruited to the site of injury and accumulate in the neointima. Expression of tissue factor (TF) by these "CD31+ myofibrocytes" is needed for progressive neointimal expansion, such that TF inhibition limits the neointima to a single layer of cells by day 28 post-injury. The aim of this study was to determine pathophysiological mediators downstream of TF that contribute to myofibrocyte-orchestrated IH. We first show that myofibrocytes make up a significant component of the neointima 28 days following injury. Using a previously defined adoptive transfer model, we then show that CD31+ myofibrocytes get recruited early to the site of injury; this model allows manipulations of the adoptively transferred cells to study how IH develops. Having confirmed that inhibition of TF on adoptively transferred cells prevents IH, we then show that TF, primarily through the generation of thrombin, induces secretion of angiopoietin-2 by myofibrocytes and this directly stimulates proliferation, inhibits apoptosis, and induces CXCL-12 production by neointimal cells, including non-fibrocytes, all of which promote progressive IH in vivo. Prior incubation to inhibit angiopoietin-2 secretion by or block TIE-2 signaling on adoptively transferred fibrocytes inhibits IH. These novel data indicate that angiopoietin-2 production by early recruited myofibrocytes critically influences the development of IH after vascular injury and suggest new therapeutic avenues for exploration.

Protective effects of Shexiang Tongxin Dropping Pill on pituitrin­induced acute myocardial ischemia in rats.

Shexiang Tongxin Dropping Pill (STP) is an established traditional Chinese medicine that is widely used for the treatment of ischemic heart disease (IHD), although its mechanisms remain unclear. The present study investigated the protective effects of STP following pituitrin (PTT)­induced myocardial ischemia in rats. ST­segment elevation, blood rheology, and the serum levels of creatine kinase­MB (CK­MB) and lactate dehydrogenase (LDH) were measured. Following heart excision, histological analysis using hematoxylin and eosin and terminal deoxynucleotidyl transferase dUTP nick end labeling were performed. The mRNA expression levels of B­cell lymphoma 2 (Bcl­2) and Bcl­2­associated X protein (Bax) were determined using reverse transcription­quantitative polymerase chain reaction, and their protein expression was detected using immunohistochemistry. The results demonstrated that STP treatment protected against ST elevation, lowered whole blood viscosity, and reduced the serum levels of CK­MB and LDH following acute myocardial ischemia. In addition, STP treatment restored the histopathological change following PTT­induced myocardial ischemia, and resulted in downregulated expression of Bax and upregulated expression of Bcl­2 in myocardial tissue. The present study demonstrates the cardioprotective ability of STP in a rat model of myocardial ischemic injury, which may be attributed to its anti­apoptotic properties. The cardioprotective properties of STP require further investigation to determine whether it may be used for the clinical treatment of IHDs.