Characteristics of human arterial smooth muscle cell cultures infected with cytomegalovirus

Summary Distinct, sequential events occurring during the destruction and simultaneous regrowth of human arterial smooth muscle cell (SMC) cultures infected with cytomegalovirus (CMV, AD169 strain) were characterized. The events were influenced by the typical phenotypic diversity reflecting relative states of differentiation of the SMC cultures. Progenitors of regeneration were a surviving population of small, undifferentiated or relatively undifferentiated SMCs. As these cells reached confluence focally, the number of cells reactive with antismooth muscle serum, i.e. differentiating, increased, and in some postconfluent foci the organization of SMCs resembled the topography of uninfected cultures. Thus, infected SMC cultures had a limited capacity to repopulate, to organize typically, and to differentiate. However, continuing cytopathic effects gradually destroyed much of the regrowth, and a relatively large, nondividing SMC with prominent cytoplasmic filaments, similar to SMCs in terminal, uninfected cultures, predominated. Infected cultures consisting overwhelmingly of the large terminal phenotype were far less productive of infectious CMV than cultures populated by SMCs with continuing capacity to divide. Gradually, cultures consisting of the terminal phenotype deteriorated as a result of sporadic cytopathic effects of CMV and an effect resembling “senescent” degeneration in uninfected, nondividing cultures in late passage. The infected, terminal phenotype could be a latent or persistent source of CMV antigen or nucleic acid-positive cells detected by different investigators in normal and in atheromatous, human tissue, assuming that it exists and survives for an extended period in vivo after infection of vascular SMC. The derivation of smooth muscle cell lines used in this investigation was supported through National Research and Demonstration Center grant HL-17269-07 from the National Heart, Lung and Blood Institute, Bethesda, MD.     

动脉平滑肌细胞原代培养贴块法的改良

对传统的动脉平滑肌细胞（ASMCs）的体外培养贴块法进行改良。用改良的贴块法进行ASMCs的原代培养,用传统的贴块培养法作对照。运用改良贴块法细胞长满瓶壁所需平均生长时间为6天,产量为60-60万/瓶;而在同等条件下对照组的传统贴块细胞长满瓶壁所需生长时间约需15天,产量仅为30万/瓶。并且,改良方法比传统方法具有较高的成功率,且无需选用胰岛素。新方法简单易行,结果稳定可靠。     

The proteome and secretome of human arterial smooth muscle cells

Smooth muscle cells (SMCs) play a crucial role in cardiovascular disorders. A differential proteomic approach should help to elucidate SMC dysfunctions involved in these diseases. With this goal in mind, we plotted the first 2-dimensional (2-D) maps of the proteome and secretome of human arterial smooth muscle cell (ASMC). Intracellular and secreted proteins were extracted from a primary culture of SMCs obtained from patients undergoing coronary artery bypass surgery (n = 11) and separated by 2-dimensional gel electrophoresis. Silver-stained gels were analyzed using Progenesis software. A high level of between-gel reproducibility was obtained, allowing us to generate two protein patterns specific to the ASMC proteome and secretome, respectively. A total of 121 and 40 distinct intracellular and secreted polypeptide spots, corresponding to 83 and 18 different proteins, respectively, were identified by matrix-assisted laser desorption/ionization mass spectrometry. The 2-D reference maps and database resulting from this study confirm that SMCs are involved in a wide range of biological functions. They could constitute a useful tool for a wide range of investigators involved in vascular biology, allowing them to investigate SMC protein changes associated with cardiovascular disorders or environmental stimuli.     

Autologous bone marrow mononuclear cell therapy improves symptoms in patients with end-stage peripheral arterial disease and reduces inflammation-associated parameters

Background aimsThe purpose of this study was to evaluate the effect of autologous bone marrow mononuclear cells (BM-MNCs) on symptoms and perfusion indices in severely symptomatic patients with peripheral arterial disease (PAD) without further option for endovascular or surgical revascularization.MethodsOnly patients with severe symptomatic PAD (Fontaine class IIb-IV, Rutherford category 3–6) not amenable for revascularization were treated. Bone marrow from both cristae iliacae was harvested; MNCs were isolated by the Ficoll density-gradient method and transplanted by means of intra-arterial and intramuscular injection in the index limb. Functional (pain score, ulcer healing, maximum walking distance) and perfusion indices such as ankle-brachial-index and transcutaneous oxygen pressure were documented before and after BM-MNC therapy. Additionally, serum concentration of C-reactive protein and interleukin-6 were measured as markers of inflammation before and after BM-MNC treatment.ResultsSixteen consecutive patients (four women; mean age, 63.0 ± 13 years) were treated with a mean dose of 4.2 ± 2.2 × 10⁸ BM-MNCs. At 6 months' follow-up, ankle-brachial-index, transcutaneous oxygen pressure and maximum walking distance significantly increased, whereas C-reactive protein and interleukin-6 conversely decreased (P < 0.01 versus baseline values), resulting in 88% limb salvage, 75% pain reduction and 71% complete wound healing and/or reduction of ulcer size. One major and one minor amputation were performed, both in patients with Rutherford category 6.ConclusionsAutologous BM-MNC therapy in patients with end-stage PAD improves tissue perfusion indices and decreases markers of inflammation. If our observations could be confirmed by large-scale, randomized controlled trials, BM-MNC transplantation could become an alternative therapeutic option for patients with end-stage PAD.     

PDGF-induced proliferation in human arterial and venous smooth muscle cells: molecular basis for differential effects of PDGF isoforms

Platelet‐derived growth factor (PDGF) has been implicated in the pathogenesis of arterial atherosclerosis and venous neointimal hyperplasia. We examined the effects of PDGF isoforms on smooth muscle cells (SMCs) from arterial and venous origins in order to further understand the differential responsiveness of these vasculatures to proliferative stimuli. Serum‐starved human arterial and venous SMCs exhibited very different proliferative responses to PDGF isoforms. Whereas, proliferation of arterial SMCs was strongly stimulated by PDGF‐AA, venous SMCs showed no proliferative response to PDGF‐AA, but instead demonstrated a significantly greater proliferative response to PDGF‐BB than arterial SMCs. Part of this difference could be attributed to differences in PDGF receptors expression. There was a 2.5‐fold higher (P < 0.05) density of PDGF receptor‐α (PDGF‐Rα) and a 6.6‐fold lower (P < 0.05) density of PDGF‐Rβ expressed on arterial compared to venous SMCs. Concomitant with an increased proliferative response to PDGF‐AA in arterial SMCs was a marked PDGF‐Rα activation, enhanced phosphorylation of ERK1/2 and Akt, a transient activation of c‐Jun NH2‐terminal kinase (JNK), and a significant reduction in expression of the cell‐cycle inhibitor p27^kip1. This pattern of signaling pathway changes was not observed in venous SMCs. No phosphorylation of PDGF‐Rα was detected after venous SMC exposure to PDGF‐AA, but there was enhanced phosphorylation of ERK1/2 and Akt in venous SMCs, similar to that seen in the arterial SMCs. PDGF‐BB stimulation of venous SMC resulted in PDGF‐Rβ activation as well as transactivation of epidermal growth factor receptor (EGF‐R); transactivation of EGF‐R was not observed in arterial SMCs. These results may provide an explanation for the differential susceptibility to proliferative vascular diseases of arteries and veins. J. Cell. Biochem. 112: 289–298, 2011. © 2010 Wiley‐Liss, Inc.     

A simple means of increasing muscle size after spinal cord injury: a pilot study

This study tested that hypothesis that skeletal muscle within a year of spinal cord injury (SCI) would respond to intermittent high force loading by showing an increase in size. Three males about 46 weeks post clinically complete SCI underwent surface electrical stimulation of their left or right m. quadriceps femoris 2 days per week for 8 weeks to evoke 4 sets of ten isometric or dynamic actions each session. Conditioning increased average cross-sectional area of m. quadriceps femoris, assessed by magnetic resonance imaging, by 20+/-1% (p = 0.0103). This reversed 48 weeks of atrophy such that m. quadriceps femoris 54 weeks after SCI was the same size as when the patients were first studied 6 weeks after injury. The results suggest that skeletal muscle is remarkably responsive to intermittent, high force loading after almost one year of little if any contractile activity.     

Role of proteomic technologies in understanding risk of arterial thrombosis

Arterial thrombosis is a pivotal event in the development of cardiovascular diseases. Plasma and cellular proteins have the potential to influence thrombus morphology and function. This review summarizes the latest studies to use proteomic technologies to characterize the cellular and plasma components involved in arterial thrombosis, with a view to understanding the pathogenesis and treatment of acute cardiovascular diseases. Proteomic approaches have been extensively used to profile the proteome of endothelial cells, leukocytes, vascular smooth muscle cells, platelets and plasma in the search for risk factors for cardiovascular disease; however, further work is required to validate the direct contribution of these proteins to arterial thrombosis.     

大鼠细小肺动脉平滑肌细胞原代培养和鉴定方法的研究

目的：建立一种重复性好、培养周期短及传代次数多的大鼠细小肺动脉平滑肌细胞（PASMCs）培养方法。方法：在无菌条件下,分离雄性SD大鼠肺细小动脉,剥离外膜和剔除内皮细胞,经胶原酶I消化,培养PASMCs。0.4%台盼蓝染色测定细胞活力;倒置相差显微镜观察;免疫细胞化学法和免疫荧光染色法,进行平滑肌α-肌动蛋白（α-SMactin）鉴定。结果：形态学观察、免疫细胞化学法及免疫荧光染色法鉴定表明培养细胞为PASMCs;细胞存活率在96.5%以上;原代培养后4～7d即可传代,并且生长特点、细胞形态不易发生改变。结论：采用胶原酶I消化法培养PASMCs,方法简单、酶消化时间易控制、培养周期短、重复性好,培养的原代PASMCs具有数量多和生长迅速的特点。     

Development of concurrent magnetic resonance imaging and volumetric optoacoustic tomography: A phantom feasibility study

Optoacoustic tomography (OAT) and magnetic resonance imaging (MRI) provide highly complementary capabilities for anatomical and functional imaging of living organisms. Herein, we investigate on the feasibility of combining both modalities to render concurrent images. This was achieved by introducing a specifically-designed copper-shielded spherical ultrasound array into a preclinical MRI scanner. Phantom experiments revealed that the OAT probe caused minimal distortion in the MRI images, while synchronization of the laser and the MRI pulse sequence enabled defining artifact-free acquisition windows for OAT. Good dynamic OAT contrast from superparamagnetic iron oxide nanoparticles, a commonly used agent for MRI contrast enhancement, was also observed. The hybrid OAT-MRI system thus provides an excellent platform for cross-validating functional readings of both modalities. Overall, this initial study serves to establish the technical feasibility of developing a hybrid OAT-MRI system for biomedical research.     

Differential effects of imatinib on PDGF-induced proliferation and PDGF receptor signaling in human arterial and venous smooth muscle cells

Platelet-derived growth factor (PDGF) has been implicated in smooth muscle cell (SMC) proliferation, a key event in the development of myointimal hyperplasia in vascular grafts. Recent evidence suggests that the PDGF receptor (PDGFR) tyrosine kinase inhibitor, imatinib, can prevent arterial proliferative diseases. Because hyperplasia is far more common at the venous anastomosis than the arterial anastomosis in vascular grafts, we investigated whether imatinib also inhibited venous SMC (VSMC) proliferation, and examined possible differences in its mechanism of action between VSMC and arterial SMC (ASMC). Human ASMC and VSMC were stimulated with PDGF-AB, in the presence or absence of imatinib (0.1-10 microM). Proliferation was assayed using the 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, while PDGFR, Akt and ERK1/2-mitogen activated protein kinase (MAPK) signaling pathways were investigated by immunoblotting. The proliferative response to PDGF at 50 and 100 ng/ml was 32 and 43% greater, respectively, in VSMC than in ASMC. Similarly, PDGF-stimulated proliferation was more sensitive to inhibition by imatinib in VSMC than ASMC (IC(50) = 0.05 microM vs. 0.4 microM; P < 0.01). Imatinib also more effectively inhibited PDGF-induced phosphorylation of PDGFRbeta and Akt in VSMC, compared to ASMC. These data highlight inherent pharmacodynamic differences between VSMC and ASMC in receptor and cell signaling functions and suggest that imatinib therapy may be useful for the prevention of venous stenosis in vascular grafts.     

Human expression patterns: qualitative and quantitative analysis of thrombospondin‐1 under physiological and pathological conditions

Thrombospondin‐1 (TSP‐1), a matricellular protein and one of the first endogenous anti‐angiogenic molecules identified, has long been considered a potent modulator of human diseases. While the therapeutic effect of TSP‐1 to suppress cancer was investigated in both research and clinical settings, the mechanisms of how TSP‐1 is regulated in cancer remain elusive, and the scientific answers to the question of whether TSP‐1 expressions can be utilized as diagnostic or prognostic marker for patients with cancer are largely inconsistent. Moreover, TSP‐1 plays crucial functions in angiogenesis, inflammation and tissue remodelling, which are essential biological processes in the progression of many cardiovascular diseases, and therefore, its dysregulated expressions in such conditions may have therapeutic significance. Herein, we critically analysed the literature pertaining to TSP‐1 expression in circulating blood and pathological tissues in various types of cancer as well as cardiovascular and inflammation‐related diseases in humans. We compare the secretion rates of TSP‐1 by different cancer and non‐cancer cells and discuss the potential connection between the expression changes of TSP‐1 and vascular endothelial growth factor (VEGF) observed in patients with cancer. Moreover, the pattern and emerging significance of TSP‐1 profiles in cardiovascular disease, such as peripheral arterial disease, diabetes and other related non‐cancer disorders, are highlighted. The analysis of published TSP‐1 data presented in this review may have implications for the future exploration of novel TSP‐1‐based treatment strategies for cancer and cardiovascular‐related diseases.     

Medial calcification in the arterial wall of smooth muscle cell‐specific Smpd1 transgenic mice: A ceramide‐mediated vasculopathy

Arterial medial calcification (AMC) is associated with crystallization of hydroxyapatite in the extracellular matrix and arterial smooth muscle cells (SMCs) leading to reduced arterial compliance. The study was performed to test whether lysosomal acid sphingomyelinase (murine gene code: Smpd1)‐derived ceramide contributes to the small extracellular vesicle (sEV) secretion from SMCs and consequently leads to AMC. In Smpd1^trg/SM^cre mice with SMC‐specific overexpression of Smpd1 gene, a high dose of Vit D (500 000 IU/kg/d) resulted in increased aortic and coronary AMC, associated with augmented expression of RUNX2 and osteopontin in the coronary and aortic media compared with their littermates (Smpd1^trg/SM^wt and WT/WT mice), indicating phenotypic switch. However, amitriptyline, an acid sphingomyelinase (ASM) inhibitor, reduced calcification and reversed phenotypic switch. Smpd1^trg/SM^cre mice showed increased CD63, AnX2 and ALP levels in the arterial wall, accompanied by reduced co‐localization of lysosome marker (Lamp‐1) with multivesicular body (MVB) marker (VPS16), a parameter for lysosome‐MVB interaction. All these changes related to lysosome fusion and sEV release were substantially attenuated by amitriptyline. Increased arterial stiffness and elastin disorganization were found in Smpd1^trg/SM^cre mice as compared to their littermates. In cultured coronary arterial SMCs (CASMCs) from Smpd1^trg/SM^cre mice, increased P_i concentrations led to markedly increased calcium deposition, phenotypic change and sEV secretion compared with WT CASMCs, accompanied by reduced lysosome‐MVB interaction. However, amitriptyline prevented these changes in P_i‐treated CASMCs. These data indicate that lysosomal ceramide plays a critical role in phenotype change and sEV release in SMCs, which may contribute to the arterial stiffness during the development of AMC.     

Optimization of extracorporeal membrane oxygenation therapy using near‐infrared spectroscopy to assess changes in peripheral circulation: A pilot study

Near‐infrared spectroscopy (NIRS) has been proposed as a noninvasive modality for detecting complications in patients undergoing extracorporeal membrane oxygenation (ECMO), and it can simultaneously reveal the global circulatory status of these patients. We optimized ECMO therapy on the basis of real‐time peripheral NIRS probing. Three patients underwent venoarterial (VA) ECMO and one patient underwent venovenous (VV) ECMO. All patients received peripheral ECMO cannulation with routine distal perfusion catheter placement. We designed an experimental protocol to adjust ECMO blood flow over 1 hour. Hemodynamic responses were measured using NIRS devices attached to the calf at approximately 60% of the distance from the ankle to the knee. HbO₂ levels change substantially with adjustments in ECMO flow, and they are more sensitive than HHb levels and the tissue saturation index (TSI) are. NIRS for optimizing ECMO therapy may be reliable for monitoring global circulatory status.     

Muscle oxygenation dynamics in response to electrical stimulation as measured with near‐infrared spectroscopy: A pilot study

Neuromuscular electrical stimulation (NMES) is used for preventing muscle atrophy and improving muscle strength in patients and healthy people. However, the current intensity of NMES is usually set at a level that causes the stimulated muscles to contract. This typically causes pain. Quantifying the instantaneous changes in muscle microcirculation and metabolism during NMES before muscle contraction occurs is crucial, because it enables the current intensity to be optimally tuned, thereby reducing the NMES‐induced muscle pain and fatigue. We applied near‐infrared spectroscopy (NIRS) to measure instantaneous tissue oxygenation and deoxygenation changes in 43 healthy young adults during NMES at 10, 15, 20, 25, 30, and 35 mA. Having been stabilized at the NIRS signal baseline, the tissue oxygenation and total hemoglobin concentration increased immediately after stimulation in a dose‐dependent manner (P < 0.05) until stimulation was stopped at the level causing muscle contraction without pain. Tissue deoxygenation appeared relatively unchanged during NMES. We conclude that NIRS can be used to determine the optimal NMES current intensity by monitoring oxygenation changes.      

Bilateral administration of autologous CD133+ cells in ambulatory patients with refractory critical limb ischemia: lessons learned from a pilot randomized,double-blind,placebo-controlled trial

Background aimsCD133+ cells confer angiogenic potential and may be beneficial for the treatment of critical limb ischemia (CLI). However, patient selection, blinding methods and end points for clinical trials are challenging. We hypothesized that bilateral intramuscular administration of cytokine-mobilized CD133+ cells in ambulatory patients with refractory CLI would be feasible and safe.MethodsIn this double-blind, randomized sham-controlled trial, subjects received subcutaneous injections of granulocyte colony-stimulating factor (10 μg/kg per day) for 5 days, followed by leukapheresis, and intramuscular administration of 50–400 million sorted CD133+ cells delivered into both legs. Control subjects received normal saline injections, sham leukapheresis and intramuscular injection of placebo buffered solution. Subjects were followed for 1 year. An aliquot of CD133+ cells was collected from each subject to test for genes associated with cell senescence.ResultsSeventy subjects were screened, of whom 10 were eligible. Subject enrollment was suspended because of a high rate of mobilization failure in subjects randomly assigned to treatment. Of 10 subjects enrolled (7 randomly assigned to treatment, 3 randomly assigned to control), there were no differences in serious adverse events at 12 months, and blinding was preserved. There were non-significant trends toward improved amputation-free survival, 6-minute walk distance, walking impairment questionnaire and quality of life in subjects randomly assigned to treatment. Successful CD133+ mobilizers expressed fewer senescence-associated genes compared with poor mobilizers.ConclusionsBilateral administration of autologous CD133+ cells in ambulatory CLI subjects was safe, and blinding was preserved. However, poor mobilization efficiency combined with high CD133+ senescence suggests futility in this approach.     

A comparative study of muscle force estimates using Huxley's and Hill's muscle model

Determination of muscle forces in individual muscles is often essential to assess optimal performance of human motion. Inverse dynamic methods based on the kinematics of the given motion and on the use of optimisation approach are the most widely used for muscle force estimation. The aim of this study was to estimate how the choice of muscle model influences predicted muscle forces. Huxley's (1957, Prog Biophys Biop Chem. 7: 255–318) and Hill's (1938, Proc R Soc B. 126: 136–195) muscle models were used for determination of muscle forces of two antagonistic muscles of the lower extremity during cycling. Huxley's model is a complex model that couples biochemical and physical processes with the microstructure of the muscle whereas the Hill's model is a phenomenological model. Muscle forces predicted by both models are within the same range. Huxley's model predicts more realistic patterns of muscle activation but it is computationally more demanding. Therefore, if the overall muscle forces are to be assessed, it is reasonable to use a simpler implementation based on Hill's model.     

A study of C-reactive protein,lipid metabolism and peripheral blood to identify a link between periodontitis and cardiovascular disease

Periodontitis is characterized by systemic inflammatory host responses that may contribute to a higher risk for cardiovascular disease. We hypothesized that periodontitis may be associated with altered C-reactive protein levels, serum levels of lipids and peripheral blood counts, and that these characteristics may serve as markers for a link between periodontitis and cardiovascular disease. Sixty subjects, 25–60 years old, were divided into three groups of 20 subjects each. Group 1, age and sex matched healthy controls; group 2, patients diagnosed with chronic periodontitis; group 3, patients diagnosed with acute periodontal lesions including periodontal abscess and pericoronal abscesses. Serum C-reactive protein levels, lipid levels and peripheral blood counts were obtained for all three groups. Significant increases in C-reactive protein and serum lipid levels, and altered peripheral blood counts were observed between the experimental groups; these factors were correlated with chronic periodontitis and cardiovascular disease. These simple, economical clinical measurements can be used to assess periodontal tissue damage and may be useful for predicting risk of cardiovascular disease in these subjects.     

A comparative study on the antioxidant effects of hesperidin and ellagic acid against skeletal muscle ischemia/reperfusion injury

Abstract

The antioxidant effects of ellagic acid (EA) and hesperidin (HES) against skeletal muscle ischemia/reperfusion injury (I/R) were performed. Hindlimb ischemia has been induced by tourniquet occlusion for 2?h on left hindlimb. At the end of ischemia, the tourniquate has been removed and initiated reperfusion for 2?h. EA (100?mg/kg) has been applied orally before ischemia/reperfusion in the EA?+?I/R group. HES (100?mg/kg) has been given orally in the HES?+?I/R group. The left gastrocnemius muscle has been harvested and stored immediately at??80?°C until assessed for the levels of MDA and antioxidant enzymes activities. MDA level has statistically increased in I/R group (p?<?0.05) compared to other groups. The muscle tissue antioxidant enzymes activities were lower than the other groups in the I/R group (p?<?0.05). EA and HES treatments significantly reversed the damage level in I/R, also activity of tissue SOD increased in the EA?+?I/R and HES?+?I/R groups.     

A comparative study of human muscle and brain creatine kinases expressed in Escherichia coli

We report the expression of the human muscle (CK-MM) and brain (CK-BB) creatine kinases in Escherichia coli. The proteins have been purified to apparent homogeneity and several of their physical and kinetic properties investigated. In the process, we have conclusively verified the correct DNA sequence of the genes encoding the respective isozymes, and determined the correct primary structure and mass of the gene products. Alignment of the primary sequences of these two enzymes shows 81% sequence identity with each other, and no obvious gross structural differences. However, Western blot analyses demonstrated the general lack of antigenic cross-reactivity between these isozymes. Preliminary kinetic analyses show the K _m and k _cat values for the creatine and MgATP substrates are similar to values reported for other isozymes from various tissues and organisms. The human muscle and brain CKs do not, however, exhibit the synergism of substrate binding that is observed, for example, in rabbit muscle creatine kinase.