The effect of the total glysosidic extract of the plant Sarcolobus globosus was investigated on the contractions of the smooth muscle of the guinea-pig ileal longitudinal muscle and taenia coli. In the ileal longitudinal muscle, addition of the extract inhibited the electrical field-stimulated twitches. Similarly to verapamil, it also reduced the contractions of the muscle to acetylcholine, histamine and KCl. However, only the tonic contraction to KCl was reversed by increasing the extracellular calcium concentration. In the taenia coli, lower concentrations of both the extract and verapamil induced a parallel displacement of the dose-response curves to calcium (0.30-30 mM). Addition of the extract also dose-dependently inhibited the KCl-induced contraction of the taenia coli. Increasing the calcium concentration increased the IC50 values of the extract. The result suggests that the inhibitory effect of the Sarcolobus globosus extract on the smooth muscle, like verapamil, is mainly due to inhibition of calcium influx.
Goniothalamin (GN), a styryl-lactone isolated from Goniothalamus andersonii, has been demonstrated to possess antirestenostic properties by inducing apoptosis on coronary artery smooth muscle cells (CASMCs). In this study, the molecular mechanisms of GN-induced CASMCs apoptosis were further elucidated. Apoptosis assessment based on the externalization of phosphatidylserine demonstrated that GN induces CASMCs apoptosis in a concentration-dependent manner. The GN-induced DNA damage occurred with concomitant elevation of p53 as early as 2 h, demonstrating an upstream signal for apoptosis. However, the p53 elevation in GN-treated CASMCs was independent of NAD(P)H: quinone oxidoreductase 1 and Mdm-2 expression. An increase in hydrogen peroxide and reduction in free thiols confirmed the role for oxidative stress in GN treatment. Pretreatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-FMK) that significantly abrogated GN-induced CASMCs apoptosis suggested the involvement of caspase(s). The role of apical caspase-2, -8, and -9 was then investigated, and sequential activation of caspase-2 and -9 but not caspase-8 leading to downstream caspase-3 cleavage was observed in GN-treated CASMCs. Reduction of ATP level and decrease in oxygen consumption further confirmed the role of mitochondria in GN-induced apoptosis in CASMCs. The mitochondrial release of cytochrome c was seen without mitochondrial membrane potential loss and was independent of cardiolipin. These data provide insight into the mechanisms of GN-induced apoptosis, which may have important implications in the development of drug-eluting stents.
In-stent restenosis is caused by the proliferation of the smooth muscle cells (SMCs) following a host response towards the implanted device. However, the precise biochemical and cellular mechanisms are still not completely understood. In this paper, the behaviour of SMCs has been investigated by an in vitro model where the cells were stimulated by platelet derived growth factor (PDGF) on tissue-like substrates as well as on biomaterials such as stainless steel (St) and diamond-like carbon (DLC)-coated St. The results demonstrated that SMCs have a completely different adhesion mode on St and become particularly prone to proliferation and pro-inflammatory cytokine secretion under PDGF stimulus. This would suggest that restenosis may caused by the accidental contact of the SMC with the St substrate under an inflammatory insult.
Breast hamartoma is an uncommon poorly recognised benign breast neoplasm. Hamartoma displaying marked smooth muscle components known as myoid hamartoma of the breast is a much rarer entity. We present a case of myoid hamartoma of breast with chondroid differentiation in a 46-year-old woman. The painless breast lump was circumscribed and mammography showed a well-encapsulated large, dense mass with no calcification. Core needle biopsy was reported as fibroadenoma. The lesion was excised. Microscopically, it composed of many groups of mammary glandular components with dense fibrous stroma, adipose tissue and marked groups of smooth muscle fibres. Foci of chondroid differentiation were noted in the lesion. The smooth muscle cells showed strong and diffuse immunoreactivity for vimentin, myogloblin, alpha-smooth muscle actin, desmin and CD34 and failed to express pan-cytokeratin or S100 protein. The ducts lined by epithelial cells were reactive to pan-cytokeratin while the myoepithelial cells were reactive to S100 protein. The various immuno-histochemical staining as well as the cyto-histological changes encountered in myoid hamartomas are discussed with clinical, radiological and pathological correlation to differentiate it from other benign and malignant breast lesions.
AIM: To study the clinical features, histology and immunohistochemical properties of gastrointestinal stromal tumours (GISTs); and establish any parameters that can help prognosticate the malignant potential.
METHODS: Twenty-six patients with GISTs who were seen in Sultanah Aminah Hospital Johor, Malaysia from 1999 to 2003 were selected for study. Patient, clinical characteristics and outcome based on surgical records were analysed. Tumour variables (tumour size, cellularity, mitotic count, necrosis and haemorrhage) were compared between very low to low risk groups and intermediate to high risk groups. The immunohistochemical properties of GISTs were also studied.
RESULTS: Patients with GISTs presented mainly with pain, palpable mass or gastrointestinal tract bleeding. The tumours were seen in stomach (50%) followed by small intestine (38.5%) and rectum (11.5%). In the period of study, six patients had metastasis, mainly in the liver or peritoneum. Immunoreactivity for CD117, CD34, vimentin, S100, neuron specific enolase, alpha-smooth-muscle-actin and desmin were observed in 100%, 76.9%, 61.5%, 46.1%, 80.8%, 11.5% and 0% of tumours respectively. The behaviour of GISTs was largely dependent on tumour size and number of mitosis. Necrosis and haemorrhage were seen in tumours with high risk potential.
A clinicohistological study of acute atherosis in molar pregnancy was undertaken. Maternal decidual vessels in currettage samples of 38 histologically confirmed complete hydatidiform moles were examined histologically for acute atherosis, recognised as fibrinoid necrosis of the smooth muscle wall with a perivascular mononuclear cell infiltrate, with or without lipophages. Acute atherosis was detected in eight of 38 cases, an incidence of 18.4%. All the patients were normotensive. The significance of acute atherosis in molar pregnancy remains to be clarified.
A study of 101 sera from 69 Malay, 14 Chinese and 18 Indian healthy adult Malaysians was undertaken to determine the frequency of antinuclear antibodies (ANA), antimitochondrial antibodies (AMA), antismooth muscle antibodies (SMA) and antiparietal cell antibodies (APCA). There were 67 females and 34 males with a mean age of 31.7 years (+/-8.6). ANA was assayed by immunofluorescence (IF) using both mouse liver and HEp-2 cell substrates. AMA, SMA and APCA were also tested by IF using composite sections from mouse liver, kidney and stomach substrates. Analysis showed 6.9% were positive for ANA at a titre of 1:40 with HEp-2 while only 1.9% were detected using mouse liver. 9.9% had detectable AMA from titres 1:10 to 1:90. None of them had detectable SMA and only 1 (0.09%) had APCA at a titre of 1:80. This study suggests that a diagnosis of an autoimmune disorder has to be cautiously made taking into consideration that autoantibodies are present in low titres in the healthy population.
Adipose tissue provides an abundant source of multipotent cells, which represent a source of cell-based regeneration strategies for urinary bladder smooth muscle repair. Our objective was to confirm that adipose-derived stem cells (ADSCs) can be differentiated into smooth muscle cells.
Transforming growth factor-β (TGF-β) is a pleiotropic growth factor implicated in the development of atherosclerosis for its role in mediating glycosaminoglycan (GAG) chain hyperelongation on the proteoglycan biglycan, a phenomenon that increases the binding of atherogenic lipoproteins in the vessel wall. Phosphorylation of the transcription factor Smad has emerged as a critical step in the signaling pathways that control the synthesis of biglycan, both the core protein and the GAG chains. We have used flavopiridol, a well-known cyclin-dependent kinase inhibitor, to study the role of linker region phosphorylation in the TGF-β-stimulated synthesis of biglycan. We used radiosulfate incorporation and SDS-PAGE to assess proteoglycan synthesis, real-time polymerase chain reaction to assess gene expression, and chromatin immunoprecipitation to assess the binding of Smads to the promoter region of GAG Synthesizing genes. Flavopiridol blocked TGF-β-stimulated synthesis of mRNA for the GAG synthesizing enzymes, and chondroitin 4-sulfotransferase (C4ST-1), chondroitin sulfate synthase-1 (ChSy-1) and TGF-β-mediated proteoglycans synthesis as well as GAG hyperelongation. Flavopiridol blocked TGF-β-stimulated Smad2 phosphorylation at both the serine triplet and the isolated threonine residue in the linker region. The binding of Smad to the promoter region of the C4ST-1 and ChSy-1 genes was stimulated by TGF-β, and this response was blocked by flavopiridol, demonstrating that linker region phosphorylated Smad can pass to the nucleus and positively regulate transcription. These results demonstrate the validity of the kinases, which phosphorylate the Smad linker region as potential therapeutic target(s) for the development of an agent to prevent atherosclerosis.
There is a need for efficient and "off-the-shelf" grafts in urethral reconstructive surgery. Currently available surgical techniques require harvesting of grafts from autologous sites, with increased risk of surgical complications and added patient discomfort. Therefore, a cost-effective and cell-free graft with adequate regenerative potential has a great chance to be translated into clinical practice. Tubular cell-free collagen grafts were prepared by varying the collagen density and fiber distribution, thereby creating a polarized low fiber density collagen graft (LD-graft). A uniform, high fiber density collagen graft (HD-graft) was engineered as a control. These two grafts were implanted to bridge a 2 cm long iatrogenic urethral defect in a rabbit model. Histology revealed that rabbits implanted with the LD-graft had a better smooth muscle regeneration compared to the HD-graft. The overall functional outcome assessed by contrast voiding cystourethrography showed patency of the urethra in 90% for the LD-graft and in 66.6% for the HD-graft. Functional regeneration of the rabbit implanted with the LD-graft could further be demonstrated by successful mating, resulting in healthy offspring. In conclusion, cell-free low-density polarized collagen grafts show better urethral regeneration than high-density collagen grafts.
PREMISES AND OBJECTIVES: Antioxidant plays an important role in preventing the progression of diabetes mellitus (DM) complications. The aim of the present study was to investigate the effect of alpha lipoic acid (ALA) supplementation on plasma lipid, oxidative stress and vascular changes in diabetic rats.
Syzygium samarangense (Family; Myrtaceae) or 'makopa', as it is commonly known, is native to Malaysia, some islands of Indonesia and to Far East in general. This study was undertaken to rationalize the use of this plant in hypermotility states of the gut. The hexane extract of S. samarangense (Ss.Hex) was found to dose-dependently (10-3000 microg/mL) relax the spontaneously contracting isolated rabbit jejunum. When tested for a possible calcium channel blocking (CCB) activity, the extract (10-1000 microg/mL) relaxed the high K+-induced contractions and also decreased the Ca++ dose-response curves in a dose-dependent manner (30-100 microg/mL), confirming the CCB activity. Four flavonoids isolated from the hexane extract were tested for a possible spasmolytic activity. All flavonoids, identified as: 2'-hydroxy-4',6'-dimethoxy-3'-methylchalcone (SS1), 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (SS2), 2',4'-dihydroxy-6'-methoxy-3'-methylchalcone (SS3) and 7-hydroxy-5-methoxy-6,8-dimethylflavanone (SS4), showed dose-dependent (10-1000 microg/mL) spasmolytic activity with SS2 being the most potent. These results indicate that the presence of compounds with spasmolytic and calcium antagonist activity may be responsible for the medicinal use of the plant in diarrhoea.
We investigated the biomechanical relationship between intraluminal pressure within small mesenteric resistance arteries, oxidant activation of PKG, Ca2+ sparks, and BK channel vasoregulation. Mesenteric resistance arteries from wild type (WT) and genetically modified mice with PKG resistance to oxidative activation were studied using wire and pressure myography. Ca2+ sparks and Ca2+ transients within vascular smooth muscle cells of intact arteries were characterized using high-speed confocal microscopy of intact arteries. Arteries were studied under conditions of varying intraluminal pressure and oxidation. Intraluminal pressure specifically, rather than the generic stretch of the artery, was necessary to activate the oxidative pathway. We demonstrated a graded step activation profile for the generation of Ca2+ sparks and also a functional "ceiling" for this pressure --sensitive oxidative pathway. During steady state pressure - induced constriction, any additional Ca2+ sensitive-K+ channel functional availability was independent of oxidant activated PKG. There was an increase in the amplitude, but not the Area under the Curve (AUC) of the caffeine-induced Ca2+ transient in pressurized arteries from mice with oxidant-resistant PKG compared with wild type. Overall, we surmise that intraluminal pressure within resistance arteries controls Ca2+ spark vasoregulation through a tightly controlled pathway with a graded onset switch. The pathway, underpinned by oxidant activation of PKG, cannot be further boosted by additional pressure or oxidation once active. We propose that these restrictive characteristics of pressure-induced Ca2+ spark vasoregulation confer stability for the artery in order to provide a constant flow independent of additional pressure fluctuations or exogenous oxidants.
Atherosclerosis commences with the trapping of low density lipoproteins (LDLs) in blood vessels by modified proteoglycans (PGs) with hyperelongated glycosaminoglycan (GAG) chains. GAG chain synthesis and growth factor mediated hyperelongation regulates the composition and size of PGs in a manner that would cause low density lipoprotein (LDLs) retention in vessel wall. Galactosaminoglycans are a class of GAGs, commonly observed on PGs. Multiple enzymes are involved in galactosaminoglycan biosynthesis. Galactosaminoglycan synthesis is regulated by various signalling pathways which are amenable to pharmacological manipulation to treat atherosclerosis. Receptor mediated signalling pathways including protein tyrosine kinase receptors (PTKRs), serine/threonine kinase receptors (S/TKRs) and G-protein coupled receptors (GPCRs) pathways regulate galactosaminoglycan synthesizing enzyme expression. Increased expression of these enzymes modify galactosaminoglycan chain structure by making them hyperelongated. This review focuses on the signalling pathways regulating the expression of genes involved in galactosaminoglycan synthesis and modification. Furthermore, there are multiple other processes for inhibiting the interactions between LDL and galactosaminoglycans such as peptide mimetics of ApoB100 and anti-galactosaminoglycan antibodies and the therapeutic potential of these strategies is also addressed.
In this study, we report the effects of a non-antioxidant flavonoid flavone on vascular reactivity in Wistar-Kyoto (WKY) rat isolated aortae. Whether flavone directly modulates vascular reactivity in spontaneously hypertensive rat (SHR) and streptozotocin-induced diabetic-WKY rat isolated aortae was also determined. Thoracic aortic rings were mounted in organ chambers and exposed to various drug treatments in the presence of flavone (10 microM) or its vehicle (DMSO), which served as control. Pretreatment with flavone enhanced relaxant effects to endothelium-dependent vasodilator acetylcholine (ACh) and attenuated contractile effects to alpha(1)-receptor agonist phenylephrine (PE) in WKY aortae compared to those observed in control aortic rings. Flavone had no effect on relaxations to ACh in WKY aortae incubated with either L-NAME or methylene blue, but enhanced relaxations to ACh in WKY aortae incubated with indomethacin or partially depolarized with KCl. Relaxations to ACh are totally abolished in both control or flavone pretreated endothelium-denuded WKY aortae. Flavone attenuated the inhibition by beta-NADH of ACh-induced relaxation in WKY aortae, but it had no significant effect on the transient contractions induced by beta-NADH nor the pyrogallol-induced abolishment of ACh-induced relaxation in WKY aortae. Flavone enhanced endothelium-independent relaxation to sodium nitroprusside (SNP) in both endothelium-intact and -denuded WKY aortae. Flavone enhanced relaxation to ACh and SNP as well as attenuated contractile effects to PE in SHR and diabetic aortae, a finding similar to that observed in normal WKY aortae. From these results, we conclude that flavone modulates vascular reactivity in normal as well as hypertensive and diabetic aortae. These effects of flavone results probably through enhanced bioactivity of nitric oxide released from the endothelium.
We have investigated the effect of indomethacin on histamine- and acetylcholine (ACh)-induced responses in the intact and denuded epithelium of guinea pig isolated tracheal smooth muscle. Epithelium removal resulted in increased responsiveness to ACh and histamine. Indomethacin (2.8 microM) enhanced the sensitivity of both intact and denuded preparations to histamine and ACh. These findings suggest that the tracheal epithelium of guinea pig plays a protective role against bronchoconstrictors, such as ACh and histamine. Furthermore, indomethacin-mediated hyperresponsiveness caused by these agonists in epithelium denuded preparations might be a reflection of removal of prostaglandin (PG) biosynthesis. A similar process of interaction in indomethacin-treated asthmatic patients (with damaged airway epithelium) might take place. The significance of these findings is discussed.
Leiomyoma is a smooth muscle tumour that can arise in any part of the body especially the uterus. Even though it is traditionally linked with hormonal influence, it can also develop in extrauterine organs with a slight female predominance. It is indistinguishable with gastrointestinal stromal tumour (GIST) histologically. We report a case of a 30-year-old gentleman who presented with a huge painful mass in the right iliac fossa. Computed tomography revealed a 10 × 10 cm homogeneous mass arising from the terminal ileum; he subsequently underwent an open right hemicolectomy. Histology showed a well-circumscribed lesion composed of interlacing bundles of smooth muscle fibres of the submucosa with positive smooth muscle actin and H-Caldesmon stains but negative for DOG-1 and CD117 (c-kit) stains which were consistent with leiomyoma. Despite its rarity, this hormone-related tumour needs to be considered regardless of gender. Immunohistochemistry is paramount as it is histologically identical to GIST.
Diabetes and hypertension are closely associated with impaired endothelial function. Studies have demonstrated that regular consumption of edible palm oil may reverse endothelial dysfunction. The present study investigates the effect of palm oil fractions: tocotrienol rich fraction (TRF), alpha-tocopherol and refined palm olein (vitamin E-free fraction) on the vascular relaxation responses in the aortic rings of streptozotocin-induced diabetic and spontaneously hypertensive rats (SHR). We hypothesize that the TRF and alpha-tocopherol fractions are able to improve endothelial function in both diabetic and hypertensive rat aortic tissue. A 1,1-diphenyl picryl hydrazyl assay was performed on the various palm oil fractions to evaluate their antioxidant activities. Endothelium-dependent (acetylcholine) and endothelium-independent (sodium nitroprusside) relaxations were examined on streptozotocin-induced diabetic and SHR rat aorta following preincubation with the different fractions. In 1-diphenyl picryl hydrazyl antioxidant assay, TRF and alpha-tocopherol fractions exhibited a similar degree of activity while palm olein exhibited poor activity. TRF and alpha-tocopherol significantly improved acetylcholine-induced relaxations in both diabetic (TRF, 88.5% +/- 4.5%; alpha-tocopherol, 87.4% +/- 3.4%; vehicle, 65.0 +/- 1.6%) and SHR aorta (TRF, 72.1% +/- 7.9%; alpha-tocopherol, 69.8% +/- 4.0%, vehicle, 51.1% +/- 4.7%), while palm olein exhibited no observable effect. These results suggest that TRF and alpha-tocopherol fractions possess potent antioxidant activities and provide further support to the cardiovascular protective effects of palm oil vitamin E. TRF and alpha-tocopherol may potentially improve vascular endothelial function in diabetes and hypertension by their sparing effect on endothelium derived nitric oxide bioavailability.
The functional responses of different overnight-stored in vitro tissues are not clearly described in any animal model. The influence of overnight storage in an animal model may vary between tissue types. We employed Sprague-Dawley rat as our animal model and investigated the functional changes of rat aorta, trachea, bronchus and bladder that were used (i) immediately after surgical removal (denoted as fresh) and (ii) after storage in aerated (95% O2, 5% CO2) Krebs-Ringer bicarbonate solution at 4 °C for 24 h (denoted as stored). The aorta ring was pre-contracted with phenylephrine, and the functional response of the tissue was investigated using isoprenaline, forskolin and carbachol. Carbachol was also used to increase the tone in trachea, bronchus rings and bladder strips. A clear reduced function of endothelium, with a minor if any effect in the smooth muscle function in rat aorta was observed after overnight storage. The contractile response of overnight-stored rat airway (trachea and bronchus) and bladder smooth muscles remained unchanged. Among all tested tissues, only bronchus showed a reduced response rate (only 40% responded) after storage. In vitro rat tissues that are stored in Krebs solution at 4 °C for 24 h can still be used to investigate smooth muscle responses, however, not endothelium-mediated responses for aorta. The influence of overnight storage on different tissues from an animal model (Sprague-Dawley rat in our study) also provides an insight in maximising the use of sacrificed animals.
Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Although 58 genomic regions have been associated with CAD thus far, most of the heritability is unexplained, indicating that additional susceptibility loci await identification. An efficient discovery strategy may be larger-scale evaluation of promising associations suggested by genome-wide association studies (GWAS). Hence, we genotyped 56,309 participants using a targeted gene array derived from earlier GWAS results and performed meta-analysis of results with 194,427 participants previously genotyped, totaling 88,192 CAD cases and 162,544 controls. We identified 25 new SNP-CAD associations (P < 5 × 10-8, in fixed-effects meta-analysis) from 15 genomic regions, including SNPs in or near genes involved in cellular adhesion, leukocyte migration and atherosclerosis (PECAM1, rs1867624), coagulation and inflammation (PROCR, rs867186 (p.Ser219Gly)) and vascular smooth muscle cell differentiation (LMOD1, rs2820315). Correlation of these regions with cell-type-specific gene expression and plasma protein levels sheds light on potential disease mechanisms.