Pharmacological approach to diabetic retinopathy

Diabetic retinopathy is the most frequent cause of legal blindness in the population of 30-to-70-year olds. Whether retinopathy appears or not depends mainly on the duration of the disease and the degree of metabolic control the patient maintains. High blood glucose values lead to important changes in cellular metabolism and the main effects of these alterations are endothelial dysfunction that sets in motion the morphological process of diabetic retinopathy. The biochemical lesions caused by prolonged hyperglycemia can be positively influenced, but usually not normalized, pharmacologically with some groups of drugs, which are now under development. This makes tight control of glycemia a key measure in preventing the onset or progression of diabetic retinopathy, together with an effective program of ophthalmologic detection and follow-up in patients with diabetes. Regarding the role of endothelial dysfunction, antiplatelet drugs have been shown to slow some aspects of the evolution of diabetic retinopathy in its initial stages, mainly a lower degree of microaneurysms. However, a new approach to controlling endothelial dysfunction shows promise, mainly through the vascular endothelial growth factor (VEGF) inhibitors. These agents may prove to be especially useful in the treatment of proliferative diabetic retinopathy. Other encouraging results have been obtained in studies of antioxidant drugs and inhibitors of the formation of advanced glycation end products. Once retinal lesions appear, preventive measures need to be redoubled, with special attention to controlling glycemia; however, it is also necessary to resort to laser photocoagulation. This intervention aims to eliminate areas of ischemia and to diminish the formation of retinal exudates. If this measure fails or if vitreous hemorrhage appears, the only remaining therapeutic measure is vitrectomy.     

Proliferative response of human and minipig smooth muscle cells after coronary angioplasty to growth factors and platelets

Objectives: Platelets aggregating at the site of angioplasty, shown to be a potent proliferative stimulus for cultured smooth muscle cells (SMC), could contribute to proliferation after angioplasty.Methods: SMC were cultivated from human aorta and restenosed coronary lesions as well as from minipig aorta and from normal and post angioplasty coronary artery segments (n=6 per source). 3H-thymidine incorporation was used as a measure of proliferation.Results: 3H-thymidine incorporation varied greatly after passage 7 in all cell lines, but was significantly higher in SMC from human coronary restenosed lesions compared to those from human aorta and minipig coronary post angioplasty segments in passage 2 (44±6.4×10³ cpm/5000 SMC vs 20±3.9 and 12.1±2.1). However, all SMC exhibited a dramatic increase of 3H-incorporation after passage 7. Growth factors stimulated 3H-thymidine incorporation either dose dependently (PDGF-BB and bFGF) or only very modestly (PDGF-AA, EGF, IGF-1). The most potent stimulation was seen with PDGF-BB, 50 ng/ml, and was 17±6% (human restenosed) and 16±8% (minipig post angioplasty) of the values observed after stimulation with 10% fetal calf serum. The most effective combination of growth factors, PDGF-BB (50 ng/ml)+bFGF (20 ng/ml)+IGF-1 (50 ng/ml), produced a 3H-thymidine incorporation of 44±10% (human restenosed) and 42±11% (minipig post angioplasty) of FCS values. Stimulation by isolated platelets was dose dependent and significantly higher: 75±19% and 70±15% of FCS values for those SMC.Conclusions: 1) SMC from all sources studied exhibit significant changes of proliferation with increasing passages, excluding the comparability of data obtained with cells in different passages. 2) Data obtained with SMC from any source might not apply for SMC from human coronary restenosed lesions. 3) Currently tested growth factors do not fully account for the proliferative effect of platelets on cultured SMC.This work contains part of the Habilitationsschrift of C. Unterberg     

The Future of Collateral Artery Research

In the event of obstructive coronary artery disease, collateral arteries have been deemed an alternative bloodsource to preserve myocardial tissue perfusion and function. Monocytes play an important role in modulating this process,by local secretion of growth factors and extracellular matrix degrading enzymes. Extensive efforts have focused on developingcompounds for augmenting the growth of collateral vessels (arteriogenesis). Nonetheless, clinical trials investigatingthe therapeutic potential of these compounds resulted in disappointing outcomes. Previous studies focused on developingcompounds that stimulated collateral vessel growth by enhancing monocyte survival and activity. The limited successof these compounds in clinical studies, led to a paradigm shift in arteriogenesis research. Recent studies have shown geneticheterogeneity between CAD patients with sufficient and insufficient collateral vessels. The genetic predispositions inpatients with poorly developed collateral vessels include overexpression of arteriogenesis inhibiting signaling pathways.New directions of arteriogenesis research focus on attempting to block such inhibitory pathways to ultimately promote arteriogenesis.Methods to detect collateral vessel growth are also critical in realizing the therapeutic potential of newly developedcompounds. Traditional invasive measurements of intracoronary derived collateral flow index remain the goldstandard in quantifying functional capacity of collateral vessels. However, advancements made in hybrid diagnostic imagingmodalities will also prove to be advantageous in detecting the effects of pro-arteriogenic compounds.     

Plasma growth factor activity and cardiac wall thickness

Abstract. Objectives. The aim of this study was to investigate the growth factor activity in plasma (GFAP) in hypertension, and the correlation of GFAP to blood pressure levels, cardiac structural changes and platelet activation at rest and during exercise. Subjects. Fifteen untreated hypertensive subjects and 15 normotensive controls were recruited from a blood pressure screening programme. Interventions. GFAP before and after 30 min of strenuous exercise was analysed as the ability of patient or control plasma to stimulate incorporation of ³H-thymidine in cultured human smooth muscle cells. M-mode echocardiography was performed and platelet activity was measured by the excretion of the urinary metabolite of thromboxane A2. Results. There were no significant differences in GFAP or platelet activation at rest or after exercise between the groups. The fractions of labelled cells were 52.6% vs. 56.6% (HT vs. NT) at rest. Septum and posterior wall end-diastolic thicknesses (PWT[D]) were significantly increased in the HT group (10.4 ± 0.3 vs. 9.2 ± 0.3 mm and 11.4 ± 0.5 vs. 10.0 ± 0.4 mm, respectively, P < 0.05). PWT(D) was significantly correlated to GFAP (r = 0.40, P = 0.04) and to blood pressure (r = 0.53, P < 0.005) but there was no correlation between blood pressure and GFAP. Conclusion. The data suggest that GFAP could play a role in the early development of cardiac hypertrophy in hypertension, but that this effect does not seem to be directly linked to blood pressure levels alone.     

Historical Aspects and Relevance of the Human Coronary Collateral Circulation

In 1669, anastomoses between the right and left coronary artery were first documented by Richard Lower ofAmsterdam. Using post-mortem imaging, a debate followed on the existence of structural inter-coronary anastomoses,which was not resolved before the first half of the 20ieth century in case of the presence of coronary artery disease(CAD), and not before the early 1960ies in case of the normal human coronary circulation by William Fulton. Functionalcoronary collateral measurements during coronary interventions were first performed only in the 1970ies, respectively inthe early 1980ies. In humans, the existence of functional coronary collaterals in the absence of CAD has not been documentedbefore 2003.Though the coronary collateral circulation has been recognized as an alternative source of blood supply to ischemic myocardium,its prognostic significance for the CAD population as a whole has been controversial until recently. The debatewas due to different populations examined (acute versus chronic CAD, varying severity of CAD), to variable definitionsof the term “prognosis”, to insufficient statistical power of the investigation with rare occurrence of prognostic endpoints,to short duration of follow-up and to blunt instruments employed for collateral assessment. Individually, it has been acknowledgedthat a well functioning collateral supply to a myocardial area at risk for necrosis reduces infarct size, preservesventricular function, prevents ventricular remodelling and aneurysm formation. Collectively, evidence has accumulatedonly recently that an extensive coronary collateral circulation is a beneficial prognosticator quoad vitam. In a recentmeta-analysis on the topic, the risk ratio to die from any cause for high vs low or absent collateralization in patients withsubacute myocardial infarction was 0.53 (95% confidence interval 0.15–1.92; p=0.335), and for patients with acute myocardialinfarction, it was 0.63 (95% confidence interval 0.29–1.39; p=0.257)¸ the relative risk to die from any cause forwell vs poorly developed collaterals in patients with stable CAD was 0.59 (95% confidence interval 0.39–0.89), p=0.012.     

血管内皮细胞生长因子调动内皮祖细胞修复动脉损伤的实验研究

目的探讨血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)调动内皮祖细胞(endothelial progeni-tor cells,EPCs)修复大鼠颈总动脉损伤的作用。方法将32只大鼠切除脾脏恢复两周后随机分为两组:VEGF组和对照组,每组16只。用2F的Fogarty球囊导管损伤大鼠左颈总动脉建立动脉损伤模型。用流式细胞仪测定大鼠外周血胎儿肝脏激酶-1+(fetal liver kinase,Flk-1+)的细胞比例。动脉损伤两周后取损伤动脉段和对侧正常动脉段,采用组织学、免疫组织化学、扫描电子显微镜和图像分析技术评价各组动脉修复的效果。结果VEGF组Flk-1+的细胞比例显著高于对照组[(38.59±3.68)%vs(13.78±1.91)%,P<0.05]。两组中相同数目的外周血单个核细胞分化为EPCs的数目,VEGF组显著高于对照组[(28.25±4.23)个vs(6.88±1.34)个,P<0.05]。动脉损伤两周后,VEGF组损伤动脉的新生内膜厚度显著低于对照组[(32.38±5.58)μmvs(76.38±5.48)μm,P<0.05]。结论VEGF具有调动EPCs修复大鼠颈总动脉损伤的作用。     

Pharmacological and Non Pharmacological Strategies in the Management of Coronary Artery Disease and Chronic Kidney Disease

Patients with advanced chronic kidney disease (CKD), including those treated with dialysis, are at high risk for the development of cardiovascular disease (CVD). CVD accounts for 45-50% of deaths among dialysis patients. Therapy of acute and chronic coronary heart disease (CHD) that is effective in the general population is frequently less effective in patients with advanced CKD. Drug therapy in such patients may require dose modification in some cases. Oral anti-platelet drugs are less effective in those with advanced CKD than in persons with normal or near normal renal function. The intravenous antiplatelet drugs eptifibatide and tirofiban both require dose reductions in patients with advanced CKD. Enoxaparin requires dose reduction in early stage CKD and is contraindicated in hemodialysis patients. Unfractionated heparin and warfarin maybe used without dose adjustment in CKD patients. Atenolol, acetbutolol and nadolol may require dose adjustments in CKD. Metoprolol and carvedilol do not. Calcium channel blockers and nitrates do not require dose adjustment, whereas ranolazine does. Angiotensin converting enzyme inhibitors and angiotensin receptor blockers may safely be used in CKD patients with close observation for hyperkalemia. The safety of spironolactone in such patients is questionable. Statins are less effective in reducing cardiovascular complication in CKD patients and their initiation is not recommended in dialysis patients. Coronary artery bypass grafting is associated with higher short-term mortality, but better long-term morbidity and mortality than percutaneous coronary interventions in patients with advanced CKD with non-ST segment ACS and chronic CHD.     

Cellular and humoral responses to antigenic subunits of Echinococcus granulosus cyst fluid in hydatid patients

The hydatid fluid antigenic subunits which stimulate the proliferative response of T cells in PBMC isolated by hydatid patients were identified. In the same subjects, the serum IgG antibody profiles were determined by immuno-blotting to compare T cell activation and IgG production. The study was carried out on 18 patients with clinically and serologically diagnosed hydatidosis and on ten healthy blood donors. To assess the proliferative response to the different antigenic subunits, sheep hydatid fluid was separated by the SDS-PAGE and the fractions were blotted onto nitrocellulose and solubilized. The comparison between immunoblotting and PBMC proliferation assay shows that the 55 and 65kDa subunits of antigen 5 and the 12kDa subunit of antigen B are the most reactive subunits in the two techniques. The 16 and 20 kDa subunits of antigen B are more reactive in PBMC proliferation assay than in immunoblotting, thus assigning a specific role to antigen B in cellular response.     

VEGF Pathways in the Lymphatics of Healthy and Diseased Heart

Cardiac lymphatic system is a rare focus of the modern cardiovascular research. Nevertheless, the growing body of evidence is depicting lymphatic endothelium as an important functional unit in healthy and diseased myocardium. Since the discovery of angiogenic VEGF‐A in 1983 and lymphangiogenic VEGF‐C in 1997, an increasing amount of knowledge has accumulated on the essential roles of VEGF ligands and receptors in physiological and pathological angiogenesis and lymphangiogenesis. Tissue adaptation to several stimuli such as hypoxia, pathogen invasion, degenerative process and inflammation often involves coordinated changes in both blood and lymphatic vessels. As lymphatic vessels are involved in the initiation and resolution of inflammation and regulation of tissue edema, VEGF family members may have important roles in myocardial lymphatics in healthy and in cardiac disease. We will review the properties of VEGF ligands and receptors concentrating on their lymphatic vessel effects first in normal myocardium and then in cardiac disease.     

Apoptosis in refractory anaemia with ringed sideroblasts is initiated at the stem cell level and associated with increased activation of caspases

Treatment with granulocyte colony-stimulating factor plus erythropoietin may improve haemoglobin levels in patients with ringsideroblastic anaemia (RARS) and reduce bone marrow apoptosis. We studied bone marrow from 10 RARS patients, two of whom were also investigated after successful treatment. Mononuclear, erythroid and CD34+ cells were analysed with regard to proliferation, apoptosis, clonogenic capacity and oncoprotein expression, in the presence or absence of Fas-agonist, Fas-blocking antibody 2 and caspase-3 inhibitor. During culture, RARS bone marrow cells showed higher spontaneous apoptosis (P < 0.05) and caspase activity (P < 0.05)) than bone marrow cells from healthy donors. Eight out of nine patients had reduced growth of erythroid colony-forming units (CFU-E) (< 10% of control) and granulocyte-macrophage CFU (CFU-GM) (< 50% of control) from CD34+ cells. Fas ligation increased apoptosis and decreased colony growth equally in RARS and controls, but caused significantly more caspase activation in RARS (P < 0.01). Fas-blocking antibody showed no significant inhibitory effect on spontaneous apoptosis or ineffective haematopoiesis, as measured using phosphatidylserine exposure, the terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labelling technique, caspase activity or clonogenic growth. Caspase inhibition reduced apoptosis, increased proliferation and enhanced erythroid colony growth from CD34+ cells in RARS, but showed no effect on normal cells. CFU-E improved > 1000% after successful treatment. Thus, erythroid apoptosis in RARS is initiated at the CD34+ level and growth factor treatment may improve stem cell function. Enhanced caspase activation at the stem cell level, albeit not mediated through endogenous activation of the Fas receptor, contributes to the erythroid apoptosis in RARS.     

Cellular Proliferation and Susceptibility to Iron Toxicity in Iron Loaded Cell Cultures

Chang cells from human liver, grown in a medium supplemented with 161 μmol/1 ferric nitrilotriacetate become iron loaded with an increase in their ferritin content and total iron content. Their viability is not impaired and they survive indefinitely in this state when subcultured at regular intervals. When the cells are grown in a confluent culture their mitotic index is reduced from 12% to less than 1%. In this state they survive normally in unsupplemented medium but when iron is added at 161 umol/1 concentration the cells die within 6 weeks. The toxic lesion appears to be induced within three weeks and is irreversible unless the cells are transferred to an iron-poor medium. Cultural conditions, particularly proliferative rate, appear to be important in determining susceptibility of the cells to iron toxicity. Cell age, hypopoxia and lysosomal enzymes have been assessed as factors that may affect susceptibility.     

Coronary and Cardiac Sensitivity to the Vasoselective Benzothiazepine-like Calcium Antagonist, Clentiazem, in Experimental Heart Failure

Recent evidence suggests that newer vasoselective dihydropyridine calcium antagonists are not cardiodepressant and may be useful in the treatment of heart failure. No data are available on the efficacy of clentiazem, a vasoselective benzothiazepine-like calcium antagonist, in this pathological condition. Therefore, our objective was to assess coronary and cardiac sensitivity to clentiazem in an experimental model of chronic heart failure (cardiomyopathic hamster, UM-X7.1, >200 day old). Left ventricular developed pressure (LVP) and coronary flow changes were assessed in isolated, perfused failing hearts and in normal Syrian hamster hearts. Clentiazem dose-response curves for both coronary dilation and negative inotropic effects were determined under control conditions and in the presence of the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine (L-NAME, 30 M), and the cyclooxygenase inhibitor, indomethacin (10 M). Baseline hemodynamics indicate a significant reduction in both LVP and coronary perfusion in failing hearts. Cardiac sensitivity to the negative inotropic effects of clentiazem were similar in normal and failing hearts (IC50 = 677 nM and 734 nM, respectively). However, the clentiazem-induced increase in coronary flow was significantly attenuated in failing hearts (EC50 = 56 ± 9 nM vs. 15 ± 3 nM in normal hearts, p < 0.01). To better characterize the reduced coronary sensitivity to clentiazem in the presence of heart failure, the contributions of the NO synthase and the cyclooxygenase pathways were evaluated. Although coronary sensitivity to clentiazem was significantly reduced in the presence of L-NAME, this attenuation was of the same magnitude in normal and failing hearts, suggesting that coronary desensitization to clentiazem in failing hearts does not involve the NO synthase pathway. Experiments carried in the presence of indomethacin indicate that the reduced coronary sensitivity to clentiazem observed in failing hearts does not involve the cyclooxygenase pathway. In conclusion, reduced coronary sensitivity to the vasoselective calcium antagonist clentiazem was observed in the failing hamster heart, while no exacerbation of clentiazem's cardiodepressant actions was present. Although the mechanisms involved in the vascular desensitization to clentiazem are still unknown, our findings may provide an additional explanation for the variable efficacy of calcium antagonists in the treatment of heart failure.     

血管内皮生长因子及其受体在肺气肿患者肺组织中的表达

目的探讨血管内皮生长因子(VEGF)及其受体2(VEGF受体2/KDR)在肺气肿患者肺组织中的表达及其与肺气肿的相关性。方法取35例行肺叶切除术患者[A组(吸烟伴肺气肿组)16例,B组(不吸烟肺功能正常组)14例,C组(吸烟但肺功能正常组)5例]的外周肺组织标本,ELISA法检测肺组织匀浆中VEGF的含量,免疫组化法检测KDR蛋白表达,RT PCR检测VEGF和KDRmRNA水平,TUNEL法检测肺泡隔细胞的凋亡。结果A组患者肺组织VEGF、KDR表达均低于B组(P<0.01),肺泡隔细胞凋亡率高于B组(P<0.01)。C组与B组相比,VEGF及KDR表达差异无统计学意义(P>0.05)。结论VEGF及KDR水平减少与肺泡隔细胞凋亡的增加可能与肺气肿的发生相关。     

撤除生长因子对间充质干细胞增殖和胞外基质表达的影响

目的:研究撤除表皮生长因子(EGF)、碱性成纤维细胞生长因子(bFGF)和血管内皮细胞生长因子(VEGF)对人脐带间充质干细胞(hUCMSCs)的增殖和胞外基质(ECM)表达的影响。方法:将第5代hUCMSCs分3组:对照组(保持上述生长因子浓度不变),半量撤除组(使生长因子浓度减半),完全撤除组(不添加生长因子)。培养30h后瑞特-姬姆萨法染色观察细胞形态,MTT法细胞计数。培养15h后,定量PCR检测BCL-2、BAX及胞外基质基因的表达情况。结果:撤除生长因子后细胞增殖减慢,形态变大,核质比减小,BCL-2/BAX比率增高,胞外基质表达多数下调。半量撤除组细胞的形态、增殖能力、胞外基质表达的改变相对较小,BCL-2/BAX比率增高更明显。结论:撤除生长因子会影响细胞增殖和胞外基质的表达,保留半量生长因子可以改善这种情况。     

Coronary hemodynamics and subendocardial perfusion distal to stenoses

We compared distal coronary hemodynamics and regional myocardial perfusion in anesthetized dogs in the presence of a single or two coronary artery stenoses in series. After application of either a single or two stenoses on the left anterior descending coronary artery, regional myocardial blood flow was measured with radioactive microspheres. Moderate degrees of single-vessel stenosis (no change in resting coronary blood flow but reduction in reactive hyperemic response of 70%) resulted in no significant change in regional myocardial perfusion at rest despite a pressure drop across the stenosis of 24 +/- 3 mm Hg. When two such stenoses were applied in series, there was a 91% decrease in reactive hyperemia, a significant reduction in resting diastolic coronary blood flow and a 51 +/- 7 mm Hg pressure drop across the two stenoses. Alone, each stenosis produced no change in regional myocardial perfusion; however, together the two stenoses resulted in a significant decrease in subendocardial blood flow and a redistribution of transmural perfusion within the ischemic zone favoring the subepicardium (endo/epi from 0.95 +/- 0.03 to 0.72 +/- 0.03). The results indicate that whereas resting subendocardial perfusion is not significantly affected by moderate degrees of a single coronary artery stenosis, multiple stenoses of the same severity may dramatically reduce subendocardial perfusion.     

巴曲酶对内皮祖细胞功能的影响及其机制的探讨

目的研究巴曲酶(DF-521)对体外培养下内皮祖细胞(EPCs)的增殖、分化、成血管能力以及NO分泌能力的影响,并探讨其可能的机制。方法从人外周血中提取单个核细胞,用流式细胞术检测细胞表型CD34、CD31、血管内皮生长因子2和血管性假血友病因子(vWF),激光共聚焦显微镜观察细胞经过FITC标记的荆豆凝集素I和DiI标记的乙酰化低密度脂蛋白双染色后的情况,由此鉴定EPCs。实验分为:DF-521的低、中、高剂量干预组和对照组。检测各组EPCs的CD31和vWF表达,观察EPCs在基质胶上的成管情况以及测定培养液中NO的浓度。透射电子显微镜鉴定分化后的细胞。结果低、中、高剂量干预组EPCs的数量、CD31、vWF表达及NO浓度均明显高于对照组(P<0.05),而且这些效应随DF-521浓度升高而增加(P<0.05)。高剂量干预组EPCs形成条梭状和管腔样结构。电子显微镜观察到怀布尔-帕拉德小体。结论体外培养务件下.DF-521促进EPCs的增殖和成血管功能,并诱导其向内皮细胞分化,改善分泌NO的能力。DF-521的这些作用可能与内皮型一氧化氮合酶有关。     

Abrogation of IL-3 Requirements and Stimulation of Hematopoietic Cell Proliferationin Vitroandin Vivoby Carboxylic Acids

ABSTRACT: Short-chain fatty acids, such as butyrate and propionate, induce fetal globin gene expression and are under clinical investigation in the β-hemoglobinopathies. Limitations of the short-chain fatty acids as therapeutics include their rapid metabolism and a tendency to induce cell growth arrest if administered for prolonged periods. In studies described here, the cellular effects of other inducers of fetal globin, phenoxyacetic acid and derivatives of short-chain fatty acids and cinnamic acids, were investigated in the human erythroid cell line K562, the IL-3 dependent multi-lineage cell line (32D), and in mice and primates. Several test compounds supported 32D cell proliferation despite a 50-fold depletion of IL-3, which resulted in growth arrest and apoptotic death in control cells. The degree of proliferation induced by certain test compounds was similar to the degree of proliferation induced by Erythropoietin and G-CSF in the cells. Eight of ten compounds induced γ globin mRNA in K562 cells. A 2.5 to 6-fold increase in reticulocytosis was observedin vivoin mice treated with two prototype compounds. Pharmacokinetic studies of three prototype compounds demonstrated millimolar plasma concentrations after single oral doses for many hours in primates. These findings identify orally bioavailable compounds which induce γ globin gene expression and hematopoietic cell proliferation through an activity which partially abrogates requirements for IL-3. Such compounds provide potential for oral therapeutics which stimulate proliferation of hematopoietic cells of multiple lineages, as well as inducing fetal globin.     

Coronary risk factors in men occupationally exposed to vibration and noise

In a group of 481 men (group A ) exposed occupationally to vibration(exceeding by four times the permissible levels in the frequencyband 32–64–125 Hz) and noise (105–116 dB),and in a group of 303 men without contact with vibration andnoise at work (reference group R) the prevalence of coronaryrisk factors was assessed, Socioeconomic status, level of occupationalphysical activity and family history of heart disease were comparablein the two groups. Mean blood pressure values and the percentage with hypertensionwere significantly higher in the exposed than in the referencegroup(P0.01). Overweight and hypertrigliceridemia occurred lessfrequently in group A than in group R(P0.01 and P0.05 respectively).The prevalence of hypercholesterolaemia and smoking habits wassimilar in both groups. The results suggest that vibration andnoise may be factors which increase the risk of coronary heartdisease.