Katarzyna Koziak

Correction of posttransplant erythrocytosis with enalapril

Erythrocytosis (i.e., elevation in red cell mass) frequently develops after renal transplantation and is associated with increased risk of thromboembolic incidents and hypertension. Because it has been reported that enalapril may induce anemia in renal allograft recipients, we have undertaken a prospective study to estimate the efficacy and safety of enalapril therapy for erythrocytosis and to establish the mechanism by which enalapril reduces red cell mass. Seventeen (12 male and 5 female) long-term renal allograft recipients with increased hematocrit value (> 55% for male and > 50% for female) and elevated red cell mass as determined with 51Cr-labeled autologous erythrocytes were treated with enalapril. After 3 months of therapy, enalapril was withdrawn and patients were observed in order to differentiate spontaneous remission of erythrocytosis from effects of enalapril therapy. After 3 months of the treatment, mean hematocrit decreased from 51.1% (range 47-56%) to 42.9% (range 37-51%; P < 0.01). Red cell mass significantly decreased during this period (from 46.7 ml/kg, range 32.5-60.7 ml/kg, to 32.9 ml/kg, range 20.1-60.1 ml/kg; P < 0.01). Serum erythropoietin levels also changed from 12.2 mIU/ml (range 1.0-33.0 mIU/ml) at baseline to 5.4 mIU/ml (range 0.7-24.2 mIU/ml; P < 0.05). During the following 3 months without enalapril treatment, an increase in hematocrit was noted, reaching 51.7% (range 46-58%; P < 0.05). No serious side effects of enalapril were observed during the study, but there was a need to reduce other hypotensive drugs in some patients. Serum creatinine did not change significantly during enalapril therapy (1.49 mg/dl, range 0.9-2.3 mg/dl, and 1.55 mg/dl, range 1.0-2.3 mg/dl; before and after 3 months of therapy, respectively). Our study proves that enalapril can be safely and effectively used to treat posttransplant erythrocytosis. The effect of enalapril on red cell mass results from reducing erythropoietin production.

Identification of Suitable Reference Genes for Real-Time PCR Analysis of Statin-Treated Human Umbilical Vein Endothelial Cells

Proper data normalization in quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) is of critical importance for reliable mRNA expression analysis. Due to a diversity in putative reference genes expression stability in different in vitro models, a validation of an internal control gene should be made for each particular tissue or cell type and every specific experimental design. A few approaches have been proposed for reference gene selection, including pair-wise comparison approach and model-based approach. In this article we have assessed the expression stability of eight putative reference genes: ACTB, B2M, GADD45A, GAPDH, HPRT1, PES1, PSMC4, YWHAZ, in human umbilical vein endothelial cells (HUVEC) treated with different statins and with TNF-α. The analysis was performed with three reference gene validation programs: geNorm, NormFinder and BestKeeper. We have shown that hypoxanthine phosphoribosyltransferase 1 gene (HPRT1) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide gene (YWHAZ) are the most stably expressed genes among the analyzed ones. Furthermore, our results show that β-actin gene (ACTB) is downregulated by statins and thus should not be used as a normalizing gene in a discussed experimental setup. A ranking of candidate reference genes stability values is provided and might serve as a valuable guide for future gene expression studies in endothelial cells. This is the first report on reference gene selection for RT-qPCR applications in statin-treated HUVEC model.

Overexpression of CD39/nucleoside triphosphate diphosphohydrolase-1 decreases smooth muscle cell proliferation and prevents neointima formation after angioplasty

Summary.  Background: Growing evidence implicates the involvement of extracellular nucleotides in the regulation of platelet, leukocyte, endothelial cell (EC) and vascular smooth muscle cell (VSMC) phenotype and function. Within the quiescent vasculature, extracellular nucleotides are rapidly hydrolyzed by CD39, the dominant endothelial nucleoside triphosphate diphosphohydrolase (NTPDase‐1). However, vascular CD39/NTPDase‐1 activity is lost in EC activated by oxidative stress or proinflammatory mediators, and upon denudation of the endothelium following balloon injury. The consequent increase in extracellular nucleotide concentrations triggers signaling events leading to prothrombotic responses and increased VSMC proliferation. Objectives: To investigate the effect of overexpressed CD39/NTPDase‐1 in injured aorta. Methods: Using adenoviral‐mediated gene transfer we expressed CD39/NTPDase‐1 in mechanically denudated rat aortas. We measured intima formation by morphometry and VSMC proliferation by the [3H]‐thymidine incorporation assay. Results: Targeted expression of CD39 in injured vessels increased NTPDase activity (from 2.91 ± 0.31 to 22.07 ± 6.7 nmols Pi mg−1 protein, 4 days after exposure to the adenovirus) and prevented the formation of neointima. The thickness of the intimal layer in injured aortas exposed to Ad‐CD39 was 26.2 ± 3.9 μm vs. 51.8 ± 6.1 μm and 64.4 ± 22.2 μm (P < 0.001) in vessels treated with Ad‐β‐gal and saline, respectively. Moreover, targeted expression of CD39/NTPDase‐1 caused a 70% (P < 0.01) decrease in proliferation of VSMC isolated from transduced rat aortas as compared with VSMC derived from control vessels. Conclusions: The presented data suggest that increasing CD39/NTPDase‐1 activity in VSMC could represent a novel therapeutic approach for the prevention of stenosis associated with angioplasty and other occlusive vascular diseases.

Molecular Mechanism for Cellular Response to β-Escin and Its Therapeutic Implications.

β-escin is a mixture of triterpene saponins isolated from the horse chestnut seeds (Aesculus hippocastanum L.). The anti-edematous, anti-inflammatory and venotonic properties of β-escin have been the most extensively clinically investigated effects of this plant-based drug and randomized controlled trials have proved the efficacy of β-escin for the treatment of chronic venous insufficiency. However, despite the clinical recognition of the drug its pharmacological mechanism of action still remains largely elusive. To determine the cellular and molecular basis for the therapeutic effectiveness of β-escin we performed discovery and targeted proteomic analyses and in vitro evaluation of cellular and molecular responses in human endothelial cells under inflammatory conditions. Our results demonstrate that in endothelial cells β-escin potently induces cholesterol synthesis which is rapidly followed with marked fall in actin cytoskeleton integrity. The concomitant changes in cell functioning result in a significantly diminished responses to TNF-α stimulation. These include reduced migration, alleviated endothelial monolayer permeability, and inhibition of NFκB signal transduction leading to down-expression of TNF-α—induced effector proteins. Moreover, the study provides evidence for novel therapeutic potential of β-escin beyond the current vascular indications.

Rat Model of Parkes Weber Syndrome

The Parkes Weber syndrome is a congenital vascular malformation, characterized by varicose veins, arterio-venous fistulas and overgrown limbs. No broadly accepted animal model of Parkes Weber syndrome has been described. We created side-to-side arterio-venous fistula between common femoral vessels with proximal non-absorbable ligature on common femoral vein limiting the enlargement of the vein diameter in Wistar rats. Contralateral limb was sham operated. Invasive blood pressure measurements in both iliac and inferior cava veins were performed in rats 30 days after fistula creation. Tight circumference and femoral bone length were measured. Histopathology and morphology of soleus muscle, extensor digitorum longus muscle, and the common femoral vessel were analyzed. 30 days following arterio-venous fistula creation, a statistically significant elevation of blood pressure in common iliac vein and limb overgrowth was observed. Limb enlargement was caused by muscle overgrowth, varicose veins formation and bone elongation. Arterio-venous fistula with proximal outflow limitation led to significant increase of femoral vein circumference and venous wall thickness. Our study indicates that the described rat model mimics major clinical features characteristic for the human Parkes Weber syndrome: presence of arterio-venous fistula, venous hypertension and dilatation, varicose veins formation, and the limb hypertrophy. We reveal that limb overgrowth is caused by bone elongation, muscle hypertrophy, and venous dilatation. The newly established model will permit detailed studies on the mechanisms underlying the disease and on the efficacy of novel therapeutic strategies for the Parkes Weber syndrome treatment.