Krzysztof Bojakowski

Smooth-muscle progenitor cells of bone marrow origin contribute to the development of neointimal thickenings in rat aortic allografts and injured rat carotid arteries.

This study indicates that circulating progenitors of bone marrow origin give rise to cells with smooth muscle-like properties during formation of neointimal thickenings in the arterial wall after allotransplantation and after balloon injury. A segment of abdominal aorta was transplanted from female F344 to male LEW rats, and the grafts were analyzed for male cells by using the SRY gene as a marker. Immunostaining demonstrated that CD45-positive leukocytes made up 35–45% of the neointimal cells during the 8-week period examined. Concurrently, up to 70% of the neointimal cells were of host origin, as shown by real-time polymerase chain reaction for the SRY gene (Y chromosome). This suggests that the neointima contained host cells also of noninflammatory character. Accordingly, many cells positive for smooth-muscle &agr;-actin were detected in this layer. To explore the possible bone marrow origin of allograft cells, female LEW rats were irradiated and substituted with bone marrow from male LEW rats. Subsequently, the animals received an aortic transplant from female F344 rats or were exposed to a balloon injury of the carotid artery. Immunostaining and real-time polymerase chain reaction confirmed the above findings, but the fractions of leukocytes and SRY-positive cells were lower in the carotids than in the allografts. Combined primed in situ labeling and immunostaining verified that not only inflammatory but also smooth muscle-like cells of male origin appeared in the vessel wall in both situations. These observations suggest that the smooth-muscle cells that participate in the development of neointimal lesions during vascular disease may, in part, originate from circulating progenitors.

Monocyte Chemoattractant Protein 1–Mediated Migration of Mesenchymal Stem Cells Is a Source of Intimal Hyperplasia

Objective—Intimal hyperplasia is considered to be a healing response and is a major cause of vessel narrowing after injury, where migration of vascular progenitor cells contributes to pathological events, including transplant arteriosclerosis. Approach and Results—In this study, we used a rat aortic–allograft model to identify the predominant cell types associated with transplant arteriosclerosis and to identify factors important in their recruitment into the graft. Transplantation of labeled adventitial tissues allowed us to identify the adventitia as a major source of cells migrating to the intima. RNA microarrays revealed a potential role for monocyte chemoattractant protein 1 (MCP-1), stromal cell–derived factor 1, regulated on activation, normal T cell expressed and secreted, and interferon-inducible protein 10 in the induced vasculopathy. MCP-1 induced migration of adventitial fibroblast cells. CCR2, the receptor for MCP-1, was coexpressed with CD90, CD44, NG2, or sca-1 on mesenchymal stem cells. In vivo experiments using MCP-1–deficient and CCR2-deficient mice confirmed an important role of MCP-1 in the formation of intimal hyperplasia in a mouse model of vascular injury. Conclusions—The adventitia is a potentially important cellular source that contributes to intimal hyperplasia, and MCP-1 is a potent chemokine for the recruitment of adventitial vascular progenitor cells to intimal lesions.

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

Summary.u2002 Background:u2002Growing 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:u2002To investigate the effect of overexpressed CD39/NTPDase‐1 in injured aorta. Methods:u2002Using 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:u2002Targeted expression of CD39 in injured vessels increased NTPDase activity (from 2.91u2003±u20030.31 to 22.07u2003±u20036.7u2003nmols Piu2003mg−1 protein, 4u2003days 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 26.2u2003±u20033.9u2003μm vs. 51.8u2003±u20036.1u2003μm and 64.4u2003±u200322.2u2003μm (Pu2003<u20030.001) in vessels treated with Ad‐β‐gal and saline, respectively. Moreover, targeted expression of CD39/NTPDase‐1 caused a 70% (Pu2003<u20030.01) decrease in proliferation of VSMC isolated from transduced rat aortas as compared with VSMC derived from control vessels. Conclusions:u2002The 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.

RCMV increases intimal hyperplasia by inducing inflammation, MCP-1 expression and recruitment of adventitial cells to intima

BackgroundCytomegalovirus (CMV) infection has been associated with accelerated transplant vasculopathy. In this study, we assessed the effects of acute rat CMV (RCMV) infection on vessel remodeling in transplant vasculopathy, focusing on allograft morphology, inflammation and contribution of adventitial cells to intimal hyperplasia.MethodsInfrarenal aorta was locally infected with RCMV and transplanted from female F344 rats to male Lewis rats. Graft samples were collected 2 and 8 weeks after transplantation and analyzed for intimal hyperplasia, collagen degradation and inflammation. Transplantation of aorta followed by transplantation of RCMV infected and labeled isogenic adventitia were performed to study migration of adventitial cells towards the intima.ResultsIntimal hyperplasia was increased threefold in infected allografts. RCMV induced apoptosis in the media, expression of matrix metalloproteinase 2, and decreased collagen deposits. Macrophage infiltration was increased in the infected allografts and resulted in increased production of MCP-1. RCMV-infected macrophages were observed in the adventitia and intima. Cells derived from infected adventitia migrated towards the intima of the allograft.ConclusionsRCMV enhances infiltration of macrophages to the allografts, and thereby increases MCP-1 production and inflammation, followed by recruitment of adventitial cells to the intima and accelerated intimal hyperplasia.

Human Cytomegalovirus Inhibits Erythropoietin Production

Anemia is a feature of CKD and a complication of renal transplantation, often caused by impaired production of erythropoietin. The kidney is a target organ for human cytomegalovirus (hCMV) in such patients, but it is not known whether hCMV effects erythropoietin production. We found that kidneys from patients with CKD were positive for hCMV protein and that blood levels of hCMV IgG inversely correlated with red blood cell count. In mice, systemic murine cytomegalovirus infection decreased serum erythropoietin levels. In human erythropoietin-producing cells, hCMV inhibited hypoxia-induced expression of erythropoietin mRNA and protein. hCMV early gene expression was responsible, as ultraviolet-inactivated virus had no effect and valganciclovir treatment showed that late gene expression was nonessential. Hypoxia-induced gene transcription is controlled by the transcription factors hypoxia-inducible transcription factor (HIF)-1α and HIF2α, which are constitutively produced but stable only under low oxygen conditions. We found that hCMV inhibited constitutive production of HIF2α mRNA. HIF2α is thought to be the master regulator of erythropoietin transcription. Single-cell analysis revealed that nuclear accumulation of HIF2α was inhibited in hCMV-infected cells, and the extent of inhibition correlated with hCMV protein expression. Our findings suggest that renal hCMV infection could induce or exacerbate anemia in patients.

Ultrasound-guided angioplasty of dialysis fistula - technique description.

Summary Endovascular procedures are commonly used for treatment of vascular pathologies. These interventions are routinely performed under angiographic control. Angioplasty is increasingly more often used for correction of dialysis fistula – especially dilatation of stenosis. We describe the technique of dialysis fistula angioplasty under ultrasound control. Benefits of this procedure include lack of nephrotoxic contrast, what is especially important in chronic kidney disease patients in pre-dialysis period. Advantages of ultrasound guidance during dialysis fistula angioplasty lead to cause more and more frequent employment of this technique.

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.

Upper limb ischaemia after formation of dialysis fistula.

UNLABELLEDnLimb ischaemia caused by formation of dialysis fistula is rare but serious complication. The severity of symptoms may vary but rest pains and necrotic lesions are observed in most advance cases. In these patients different invasive procedures for treatment are performed - from simplest dialysis fistula ligation to complicated vascular reconstructions. The aim of the study was to evaluate treatment results of upper limb ischaemia triggered by dialysis fistula.nnnMATERIAL AND METHODSnWe have analysed methods and results of treatment of 14 patients with symptomatic upper limb ischaemia caused by dialysis fistula treated in our department between 1st January, 2006 and 30th June, 2013. Treatment was subject to anatomical situation and clinical symptoms. In three patients the ligation of dialysis fistula was performed, four patients underwent inflow reconstruction - in one case by ligation of ligation of vein branch, in three patients by cephalic transfer of arterial anastomosis. In 2 patients hyperkinetic fistula aneurysm was excised and replaced by PTFE bypass, in three patients fistula reconstruction with DRIL method (distal revascularization - interval ligation) was performed, in one patient surgical operation of brachial artery stenosis was conducted. One patient underwent brachial artery angioplasty.nnnRESULTSnRest pains occurred in all patients (100%), regressive changes in 10 patients (71.4%). Eight patients (57.2%) had concomitant diabetes, seven (50%) ischaemic heart disease, five (35.5%) chronic lower limb ischemia and hyperparathyroidism was observed in fivepatients (35.5%). The imaging studies in all patients revealed pathological steal syndrome (stealing blood to the fistula), in majority concurrent with other pathologies - obstruction stenosis of peripheral artery, defects in blood out flow from the limb. As a result of the surgical treatment, symptoms of limb ischaemia subsided in all patients.nnnCONCLUSIONSnCritical limb ischaemia caused by dialysis fistula is a dangerous complication. In most cases there are several causes of ischaemia. Treatment methods should be selected individually for each patient and clinical situation. Clinical symptoms should subside as a result of optimal choice of treatment and, if possible, maintaining of dialysis access.