SM22α deficiency: promoting vascular fibrosis via SRF-SMAD3-mediated activation of Col1a2 transcription following arterial injury.

Vascular fibrosis, characterized by increased Type I collagen expression, significantly contributes to vascular remodeling. Our previous studies show that disrupting the expression of SM22α (aka SM22, Tagln) induces extensive vascular remodeling following arterial injury, involving oxidative stress, inflammation, and chondrogenesis within the vessel wall. This study aims to investigate the molecular mechanisms underlying the transcription of Col1a2, a key fibrotic extracellular matrix marker. We observed upregulation of COL1A2 in the arterial wall of Sm22-/- mice following carotid injury. Bioinformatics and molecular analyses reveal that Col1a2 transcription depends on a CArG box in the promoter, activated synergistically by SRF and SMAD3. Notably, we detected enhanced nuclear translocation of both SRF and SMAD3 in the smooth muscle cells of the injured carotid artery in Sm22-/- mice. These findings demonstrate that SM22 deficiency regulates vascular fibrosis through the interaction of SRF and the SMAD3-mediated canonical TGF-ß1 signal pathway, suggesting SM22α as a potential therapeutic target for preventing vascular fibrosis.

Preparation of sustainable mineral oil-free offset printing ink with vegetable oil esters.

Mineral oils are used in substantial quantities for the production of varnishes and inks due to their abundance and versatility. However, as part of the production process, some of mineral oil components are separated as waste material, whereupon they can mix with air, water, or soil and become potentially harmful to the environment. Almost all these waste materials are volatile organic compounds (VOCs), chemicals that can easily evaporate at room temperature and have toxic effect. Therefore, a novel green, mineral oil-free offset printing ink was produced using vegetable oil esters as bio-renewable raw materials. Accompanying varnishes were prepared with linseed oil, methyl oleate, octyl stearate, and four types of resin (A, B, C, and D). The application of these varnishes to magenta color offset ink was subsequently studied to screen out the best combination of resin and ester in terms of setting time. Meanwhile, dyeing force tests were conducted to evaluate the ink's printability, while rheological analysis was done via viscosity and flowability tests. The setting time of the magenta color offset ink made by varnish A was observed to be considerably shorter than that of the ink samples prepared using varnishes B, C, and D. Furthermore, varnish A proved to be a good alternative varnish for the production of yellow, cyan, and black color offset printing inks. Samples of these inks were printed on coated paper, and their printability was contrasted against that of vegetable oil-based (pure vegetable oil), mineral oil-based, and other mineral oil-free offset printing inks. Results determined that the varnishes produced with linseed oil, methyl oleate, and octyl stearate can replace mineral oil-based varnishes for the production of offset printing ink.

Flotation de-inking for recycling paper: contrasting the effects of three mineral oil-free offset printing inks on its efficiency.

Paper for recycling has become a promising raw material for the pulp and paper industry due to its low cost and because it is conducive to sustainable development. Unfortunately, recycled paper contains a high volume of printed paper that is difficult to deink, which restricts its applications. Flotation deinking plays an essential role in the product quality and process cost of wastepaper recycling. This study was performed to evaluate the deinkability of environmentally friendly offset inks by flotation deinking. For this purpose, three mineral oil free series of four-color inks, namely, hybrid light emitting diode ultraviolet (LED-UV), LED-UV, and vegetable oil-based inks, were printed on white lightweight coated papers under laboratory conditions. The deinking methodology involves repulping, deinking agent treatment, flotation, hand sheet making, and evaluation of the produced hand sheets. The obtained results indicated that the hybrid LED-UV prints had the best deinkability. After flotation deinking, the deinking efficiency and the whiteness of the hybrid LED-UV ink increased by 58.1% and 47.6%, respectively. LED-UV ink had a 46.9% increase in the deinking efficiency and a 37.0% increase in the whiteness of the hand sheet. The deinking efficiency of the vegetable oil-based ink was the lowest, at 42.1%, and the whiteness of the hand sheet increased only by 23.8%. The particle size distribution analysis demonstrated that the hybrid LED-UV four-color ink exhibited a larger value of the average particle size than the two other. Scanning electron microscopy revealed that the hybrid LED-UV ink particles on the surface of the fibers were the least abundant after deinking. The physical strength properties of the hand sheets, including tensile index, folding resistance, and interlayer bonding strength of the hybrid LED-UV, LED-UV inks, and vegetable oil-based inks, increased.

SM22α suppresses cytokine-induced inflammation and the transcription of NF-κB inducing kinase (Nik) by modulating SRF transcriptional activity in vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) phenotypic modulation is characterized by the downregulation of SMC actin cytoskeleton proteins. Our published study shows that depletion of SM22α (aka SM22, Transgelin, an actin cytoskeleton binding protein) promotes inflammation in SMCs by activating NF-κB signal pathways both in cultured VSMCs and in response to vascular injury. The goal of this study is to investigate the underlying molecular mechanisms whereby SM22 suppresses NF-κB signaling pathways under inflammatory condition. NF-κB inducing kinase (Nik, aka MAP3K14, activated by the LTßR) is a key upstream regulator of NF-κB signal pathways. Here, we show that SM22 overexpression suppresses the expression of NIK and its downstream NF-κB canonical and noncanonical signal pathways in a VSMC line treated with a LTßR agonist. SM22 regulates NIK expression at both transcriptional and the proteasome-mediated post-translational levels in VSMCs depending on the culture condition. By qPCR, chromatin immunoprecipitation and luciferase assays, we found that Nik is a transcription target of serum response factor (SRF). Although SM22 is known to be expressed in the cytoplasm, we found that SM22 is also expressed in the nucleus where SM22 interacts with SRF to inhibit the transcription of Nik and prototypical SRF regulated genes including c-fos and Egr3. Moreover, carotid injury increases NIK expression in Sm22-/- mice, which is partially relieved by adenovirally transduced SM22. These findings reveal for the first time that SM22 is expressed in the nucleus in addition to the cytoplasm of VSMCs to regulate the transcription of Nik and its downstream proinflammatory NF-kB signal pathways as a modulator of SRF during vascular inflammation.

Hepatic artery-infusion chemotherapy improved survival of hepatocellular carcinoma after radical hepatectomy.

OBJECTIVE: To evaluate the effect of postoperative hepatic artery-infusion chemotherapy (HAIC) on survival probability in patients with hepatocellular carcinoma (HCC) after radical hepatectomy. PATIENTS AND METHODS: This retrospective study included 85 patients with HCC who received radical hepatectomy from May 2005 to May 2010. Among these patients, 42 underwent two sessions of HAIC (5-fluoruracil [1,000 mg/m2], oxaliplatin [85 mg/m2], and mitomycin-C [6 mg/m2]) after radical hepatectomy (HAIC group), and 43 underwent radical hepatectomy only (the control group). HAIC-related side effects and long-term survival were retrospectively analyzed. RESULTS: The HAIC group showed a significantly higher 5-year intrahepatic recurrence-free survival probability and lower risk of intrahepatic recurrence (HR 0.5615, 95% CI 0.3234-0.9749 [log-rank test]; P=0.0332). The HAIC group also had significantly higher 5-year disease-free survival probability (HR 0.591, 95% CI 0.3613-0.9666 [log-rank test]; P=0.0298) and overall survival probability than the control group (HR 0.5768, 95% CI 0.3469-0.9589 [log-rank test]; P=0.0278). No HAIC-related deaths in the HAIC group were reported. All toxicities and complications were controlled, and no patients quit the treatment. CONCLUSION: HAIC can effectively and safely reduce intrahepatic recurrence and improve the long-term survival of patients with HCC after radical hepatectomy.

Xiaozhi decoction reduced posthemorrhoidectomy pain and analgesic medication consumption: a prospective study.

OBJECTIVE: The aim of this study was to investigate the effect of Xiaozhi decoction (XZD) on posthemorrhoidectomy pain and analgesic medication consumption. METHODS: From May 2013 to March 2015, 315 patients who underwent open hemorrhoidectomy in our hospital were enrolled in this study, of whom, 160 patients were randomly assigned to accept sitz bath with warm water after hemorrhoidectomy (control group) and 155 patients were randomly assigned to accept sitz bath with XZD (XZD group) after hemorrhoidectomy. Postoperative pain at 12 hours after surgery and on postoperative days (PODs) 1, 2, 7, 14 and 28 was evaluated by Visual Analog Scale (VAS). Pain on defecation on PODs 1, 2, 7, 14 and 28 was also recorded using the VAS. The consumption of analgesics was also analyzed. RESULTS: No significant difference was found in baseline characteristics between the two groups. Postoperative pain score of the XZD group was significantly lower on POD 2 (6.04±1.11 vs 6.33±1.14, P=0.0229), POD 7 (3.35±0.75 vs 4.22±0.87, P=0.0000) and POD 14 (2.87±0.64 vs 3.64±0.77, P=0.0000) than that of the control group. Similarly, patients in the XZD group experienced significantly less pain on defecation on POD 2 (5.02±1.34 vs 5.43±1.56, P=0.0130), POD 7 (3.08±1.17 vs 3.52±1.29, P=0.0017) and POD 14 (2.31±0.85 vs 2.68±0.99, P=0.0004). Patients in the XZD group consumed significantly less analgesic medication on POD 2 (P=0.0136), POD 7 (P=0.0074) and POD 14 (P=0.0046) than the control group. CONCLUSION: XZD could effectively relieve postoperative pain and reduce analgesic medication consumption after hemorrhoidectomy.

SMAD3 deficiency promotes vessel wall remodeling, collagen fiber reorganization and leukocyte infiltration in an inflammatory abdominal aortic aneurysm mouse model.

TGF-ß signaling plays critical roles in the pathogenesis of aneurysms; however, it is still unclear whether its role is protective or destructive. In this study, we investigate the role of SMAD3 in the pathogenesis of calcium chloride (CaCl2)-induced abdominal aortic aneurysms (AAA) in Smad3(-/-), Smad3(+/-) and Smad3(+/+) mice. We find that loss of SMAD3 drastically increases wall thickening of the abdominal aorta. Histological analyses show significant vessel wall remodeling with elastic fiber fragmentation. Remarkably, under polarized light, collagen fibers in the hyperplastic adventitia of Smad3(-/-) mice show extensive reorganization accompanied by loosely packed thin and radial collagen fibers. The expressions of matrix metalloproteinases including MMP2, MMP9, and MMP12 and infiltration of macrophage/T cells are drastically enhanced in the vascular wall of Smad3(-/-) mice. We also observe marked increase of NF-κB and ERK1/2 signaling as well as the expression of nuclear Smad2, Smad4 and TGF-ß1 in the vessel wall of Smad3(-/-) mice. In addition, we find that SMAD3 expression is reduced in the dedifferentiated medial smooth muscle-like cells of human AAA patients. These findings provide direct in vivo evidence to support the essential roles of SMAD3 in protecting vessel wall integrity and suppressing inflammation in the pathogenesis of AAAs.

Acute porphyria in a patient with Arnold Chiari malformation.

BACKGROUND: Acute porphyria and Arnold Chiari malformation are both uncommon genetic disorders without known association. The insidious onset, non-specific clinical manifestations, and precipitating factors often cause diagnosis of acute porphyria to be missed, particularly in patients with comorbidities. CASE REPORT: A women with Arnold Chiari malformation type II who was treated with oxybutynin and antibiotics, including Bactrim for neurogenic bladder and recurrent urinary tract infection, presented with non-specific abdominal pain, constipation, and diarrhea. After receiving Flagyl for C. difficile colitis, the patient developed psychosis, ascending paralysis, and metabolic derangements. She underwent extensive neurological workup due to her congenital neurological abnormalities, most of which were unremarkable. As a differential diagnosis of Guillain Barré syndrome, acute porphyria was then considered and ultimately proved to be the diagnosis. After hematin administration and intense rehabilitation, the patient slowly recovered from the full-blown acute porphyria attack. CONCLUSIONS: This case report, for the first time, documents acute porphyria attack as a result of a sequential combination of 3 common medications. This is the first case report of the concomitant presence of both acute porphyria and Arnold Chiari malformation, 2 genetic disorders with unclear association.

SOX9 and myocardin counteract each other in regulating vascular smooth muscle cell differentiation.

Transdifferentiation of vascular smooth muscle cells (VSMC) into chondrogenic cells contributes significantly to vascular calcification during the pathogenesis of atherosclerosis. However, the transcriptional mechanisms that control such phenotypic switch remain unclear. This process is characterized by the induction of Sox9 and Col2a1 genes accompanied by the repression of myocardin (Myocd) and SMC differentiation markers such as SM22, SM α-actin and SM-MHC. Here we explore the regulatory role of SOX9, the master regulator for chondrogenesis, in modulating SMC marker gene expression. qRT-PCR and luciferase assays show that over-expression of SOX9 inhibits SMC gene transcription and promoter activities induced by myocardin, the master regulator of smooth muscle differentiation. Such suppression is independent of the CArG box in the SMC promoters but dependent on myocardin. EMSA assay further shows that SOX9 neither participates in SRF (serum response factor) binding to the CArG box nor interacts with SRF, while co-immunoprecipitation demonstrates an association of SOX9 with myocardin. Conversely, myocardin suppresses SOX9-mediated chondrogenic gene Col2a1 expression. These findings provide the first mechanistic insights into the important regulatory role of SOX9 and myocardin in controlling the transcription program during SMC transdifferentiation into chondrocytes.

Regional expression of HOXA4 along the aorta and its potential role in human abdominal aortic aneurysms.

BACKGROUND: The infrarenal abdominal aorta exhibits increased disease susceptibility relative to other aortic regions. Allograft studies exchanging thoracic and abdominal segments showed that regional susceptibility is maintained regardless of location, suggesting substantial roles for embryological origin, tissue composition and site-specific gene expression. RESULTS: We analyzed gene expression with microarrays in baboon aortas, and found that members of the HOX gene family exhibited spatial expression differences. HOXA4 was chosen for further study, since it had decreased expression in the abdominal compared to the thoracic aorta. Western blot analysis from 24 human aortas demonstrated significantly higher HOXA4 protein levels in thoracic compared to abdominal tissues (P < 0.001). Immunohistochemical staining for HOXA4 showed nuclear and perinuclear staining in endothelial and smooth muscle cells in aorta. The HOXA4 transcript levels were significantly decreased in human abdominal aortic aneurysms (AAAs) compared to age-matched non-aneurysmal controls (P < 0.00004). Cultured human aortic endothelial and smooth muscle cells stimulated with INF-γ (an important inflammatory cytokine in AAA pathogenesis) showed decreased levels of HOXA4 protein (P < 0.0007). CONCLUSIONS: Our results demonstrated spatial variation in expression of HOXA4 in human aortas that persisted into adulthood and that downregulation of HOXA4 expression was associated with AAAs, an important aortic disease of the ageing population.

Arterial injury promotes medial chondrogenesis in Sm22 knockout mice.

AIMS: Expression of SM22 (also known as SM22alpha and transgelin), a vascular smooth muscle cells (VSMCs) marker, is down-regulated in arterial diseases involving medial osteochondrogenesis. We investigated the effect of SM22 deficiency in a mouse artery injury model to determine the role of SM22 in arterial chondrogenesis. METHODS AND RESULTS: Sm22 knockout (Sm22(-/-)) mice developed prominent medial chondrogenesis 2 weeks after carotid denudation as evidenced by the enhanced expression of chondrogenic markers including type II collagen, aggrecan, osteopontin, bone morphogenetic protein 2, and SRY-box containing gene 9 (SOX9). This was concomitant with suppression of VSMC key transcription factor myocardin and of VSMC markers such as SM α-actin and myosin heavy chain. The conversion tendency from myogenesis to chondrogenesis was also observed in primary Sm22(-/-) VSMCs and in a VSMC line after Sm22 knockdown: SM22 deficiency altered VSMC morphology with compromised stress fibre formation and increased actin dynamics. Meanwhile, the expression level of Sox9 mRNA was up-regulated while the mRNA levels of myocardin and VSMC markers were down-regulated, indicating a pro-chondrogenic transcriptional switch in SM22-deficient VSMCs. Furthermore, the increased expression of SOX9 was mediated by enhanced reactive oxygen species production and nuclear factor-κB pathway activation. CONCLUSION: These findings suggest that disruption of SM22 alters the actin cytoskeleton and promotes chondrogenic conversion of VSMCs.

Resveratrol induces p53 and suppresses myocardin-mediated vascular smooth muscle cell differentiation.

Resveratrol (RSVL), a polyphenolic antioxidant present in red wine, has been shown to provide cardiovascular protection by improving endothelial function and reducing myocardial ischemia. However, little is known about how RSVL affects vascular smooth muscle cells (VSMCs) differentiation. RSVL blocks VSMC proliferation in vitro and neointimal formation following artery injury in vivo. Thus, one might expect that RSVL will promote VSMC differentiation. Unexpectedly, our results in this study show that RSVL induces VSMCs phenotypic modulation; this is characterized by suppressed transcription of SMC-specific marker genes Tagln, Acta2, Myh11, and Smtn in a dose-dependent and time-dependent manner in cultured VSMCs. Consistent with previous studies, RSVL induces the nuclear translocation of p53 and the expression of p53-responsive genes such as Cdkn1a, Gadd45a, Gadd45, and Fas. In an effort to identify the molecular mechanisms whereby RSVL represses VSMC differentiation, we found that RSVL inhibits the transcription of Myocardin (myocd) and Srf, the key VSMC transcriptional regulators. However, knockingdown and overexpressing p53 did not affect RSVL-induced VSMCs phenotypic modulation: this suggests that RSVL may induce VSMC dedifferentiation via p53-independent mechanisms. This study provides the first evidence showing that RSVL induces VSMC dedifferentiation by regulating Myocardin and SRF-mediated VSMC gene transcription.

Disruption of SM22 promotes inflammation after artery injury via nuclear factor kappaB activation.

RATIONALE: SM22 (or transgelin), an actin-binding protein abundant in vascular smooth muscle cells (VSMCs), is downregulated in atherosclerosis, aneurysm and various cancers. Abolishing SM22 in apolipoprotein E knockout mice accelerates atherogenesis. However, it is unclear whether SM22 disruption independently promotes arterial inflammation. OBJECTIVE: To investigate whether SM22 disruption directly promotes inflammation on arterial injury and to characterize the underlying mechanisms. METHODS AND RESULTS: Using carotid denudation as an artery injury model, we showed that Sm22 knockout (Sm22(-/-)) mice developed enhanced inflammatory responses with higher induction of proinflammatory genes, including Vcam1, Icam1, Cx3cl1, Ccl2, and Ptgs2. Higher expression of these genes was confirmed in primary Sm22(-/-) VSMCs and in PAC1 cells after Sm22 knockdown, whereas SM22 recapitulation in primary Sm22(-/-) VSMCs decreased their expression. NFKB2 was prominently activated in both injured carotids of Sm22(-/-) mice and in PAC1 cells after Sm22 knockdown and may mediate upregulation of these proinflammatory genes. As a NF-kappaB activator, reactive oxygen species (ROS) increased in primary Sm22(-/-) VSMCs and in PAC1 cells after Sm22 knockdown. ROS scavengers blocked NF-kappaB activation and induction of proinflammatory genes. Furthermore, Sm22 knockdown increased Sod2 expression and activated p47phox, reflecting contributions of mitochondria and NADPH oxidase to the augmented ROS production; this may result from actin and microtubule cytoskeletal remodeling. CONCLUSIONS: Our findings show that SM22 downregulation can induce proinflammatory VSMCs through activation of ROS-mediated NF-kappaB pathways. This study provides initial evidence linking VSMC cytoskeleton remodeling with arterial inflammation.

Disruption of actin cytoskeleton mediates loss of tensile stress induced early phenotypic modulation of vascular smooth muscle cells in organ culture.

Aorta organ culture has been widely used as an ex vivo model for studying vessel pathophysiology. Recent studies show that the vascular smooth muscle cells (VSMCs) in organ culture undergo drastic dedifferentiation within the first few hours (termed early phenotypic modulation). Loss of tensile stress to which aorta is subject in vivo is the cause of this early phenotypic modulation. However, no underlying molecular mechanism has been discovered thus far. The purpose of the present study is to identify intracellular signals involved in the early phenotypic modulation of VSMC in organ culture. We find that the drastic VSMC dedifferentiation is accompanied by accelerated actin cytoskeleton dynamics and downregulation of SRF and myocardin. Among the variety of signal pathways examined, increasing actin polymerization by jasplakinolide is the only one hindering VSMC dedifferentiation in organ culture. Moreover, jasplakinolide reverses actin dynamics during organ culture. Latrunculin B (disrupting actin cytoskeleton) and jasplakinolide respectively suppressed and enhanced the expression of VSMC markers, SRF, myocardin, and CArG-box-mediated SMC promoters in PAC1, a VSMC line. These results identify actin cytoskeleton degradation as a major intracellular signal for loss of tensile stress-induced early phenotypic modulation of VSMC in organ culture. This study suggests that disrupting actin cytoskeleton integrity may contribute to the pathogenesis of vascular diseases.