mTOR inhibitors and calcineurin inhibitors do not affect adhesion molecule expression of human macro- and microvascular endothelial cells.

We examined the effect of cyclosporin A, tacrolimus, sirolimus and everolimus on the cell growth, viability, proliferation, expression of cellular adhesion molecules (CAM) and leukocyte (PBMC) binding of human macrovascular (coronary artery, saphenous vein) and microvascular endothelial cells (EC). Tacrolimus did not affect EC integrity, growth or expression of CAM. Exclusively, EC from the coronary arteries showed a reduced cellular growth (about 30%) under cyclosporin A and tacrolimus treatment. In contrast, treatment with mTOR inhibitors reduced EC proliferative activity by about 40%, independently of the EC origin. No induction of apoptosis (caspase-3/7 activity) or cytotoxicity (MTS test) was observed. Long-term treatment with high concentrations of sirolimus and everolimus did not enhance the expression of CAM. Stimulation with tumor necrosis factor significantly increased the expression of CAM, independently of the drugs used. None of the mTOR inhibitors influenced the tumor necrosis factor-induced expression of CAM, whereas adhesion of PBMC increased significantly, as described by other papers. In summary, neither calcineurin inhibitors nor mTOR inhibitors activate human micro- and macrovascular EC. Therefore, the investigated drugs are unlikely to contribute to EC activation during transplant-associated vasculopathy.

Endothelial cell dysfunction after coronary artery bypass grafting with extracorporeal circulation in patients with type 2 diabetes mellitus.

OBJECTIVE: Type 2 diabetes mellitus is a well-known risk factor in patients with severe coronary artery disease undergoing coronary artery bypass grafting (CABG). The aim of the study was to analyze the endothelial dysfunction in these patients by evaluating postoperative soluble inflammatory cytokines. METHODS: Patients undergoing CABG without (n=15, group A) and with (n=14, group B) diabetes mellitus were analyzed for their release of E-selectin, interleukin-6 (IL-6), and tumor necrosis factor (TNF) up to 3 days postoperatively. A pharmacokinetic quantitative kinetic evaluation (Kinetica 2000) of maximum concentrations (c(max)), time to reach c(max) (t(max)), area under the curve (AUC(0-inf)), and terminal elimination half time (t(1/2)) was performed using a non-compartmental model. RESULTS: There was no difference in preoperative plasma concentrations of the cytokines and in the postoperative kinetic analyses of TNF when comparing both groups. However, the release of IL-6 was restricted with c(max) of 1055+/-543 pg/ml for group B versus 2112+/-1532 pg/ml for group A (p< or =0.05), paralleled by a decrease in the absolute amount (AUC(0-inf)) of IL-6. The t(1/2) remained unaffected (13.9+/-6.6h and 12.7+/-4.6h, respectively). The AUC(0-inf) of E-selectin decreased by a factor of 1.7 (p< or =0.05) with unchanged c(max) but reduced t(1/2) (12.9+/-10h for group B vs 33.1+/-20.4h for group A; p< or =0.01) referring to an augmented endothelial uptake and degradation of E-selectin. CONCLUSIONS: CABG with extracorporeal circulation could be used to verify a specific endothelial dysfunction in diabetic patients characterized by an impaired release of IL-6 and an increased turnover of E-selectin.

Impact of diabetic serum on endothelial cells: an in-vitro-analysis of endothelial dysfunction in diabetes mellitus type 2.

Diabetic endothelial dysfunction was characterized by altered levels of adhesion molecules and cytokines. Aim of our study was to evaluate the effects of diabetic serum on cell-growth and proinflammatory markers in human saphenous vein endothelial cells (HSVEC) from diabetic and non-diabetic patients. Diabetic serum showed (1) complementary proliferative activity for non-diabetic and diabetic HSVEC, (2) unchanged surface expression of adhesion molecules, and (3) elevated levels of sICAM-1 in HSVEC of all donors. The concentration of sVCAM-1 was increased only in diabetic cells. The proinflammatory state of diabetic HSVEC characterized by increased levels of cytokines was compensated. We concluded that even under normoglycemic conditions the serum itself contains critical factors leading to abnormal regulation of inflammation in diabetics. We introduced an in vitro model of diabetes representing the endothelial situation at the beginning of diabetes (non-diabetic cells/diabetic serum) as well as the diabetic chronic state (diabetic cells/diabetic serum).

Hyperglycemia increases the levels of vascular cellular adhesion molecule-1 and monocyte-chemoattractant-protein-1 in the diabetic endothelial cell.

Hyperglycemia is the major cause of diabetic angiopathy. Aim of our study was to evaluate the impact of high glucose on cell growth and function of human "diabetic" endothelial cells (EC). Incubation of non-diabetic EC with glucose moderately inhibited cell growth and increased the expression of ICAM-1 and E-selectin. In the disease-specific EC, glucose treatment resulted also in moderately inhibited cell growth by 5-10%, increased basal expression of VCAM-1 by 10-20%, and an enhanced release of monocyte-chemoattractant-protein-1 (MCP-1) by 40-70%. The expression of ICAM-1 and E-selectin and the release of IL-6 and IL-8 was not affected. The usage of our disease-specific EC model might evaluate the impact of systemic factors of diabetic patients in the progression of endothelial dysfunction, and may be suitable to develop relevant therapeutic regimens.

Validity of a patient-derived system of tissue-specific human endothelial cells: interleukin-6 as a surrogate marker in the coronary system.

The aim of our study was to evaluate the relevance of tissue- and species-specific endothelial cells (EC) to study EC-dependent mechanisms in inflammatory-mediated tissue injury. We established an isolation protocol for highly purified EC (pEC) preparations of different origin and compared EC-specific inflammatory responses. Fluorescence-activated cell separation was used to obtain pEC cultures from different human arterial (coronary artery, internal thoracic artery) and venous (umbilical vein, saphenous vein) vessels. All pEC were analyzed for growth kinetics, morphology, release of cytokines/chemokines, and expression of E-selectin. For all different EC cultures, purities of >or=99% were reproducibly achieved. The EC isolation did not affect EC growth, morphology, and function. However, characterization of pEC from different vessel materials revealed an intrinsic, tissue-specific functional heterogeneity of EC cultures. Despite an arterial and venous difference in the secretion of IL-8 and monocyte chemoattractant protein-1, especially EC from coronary arteries produced significantly more IL-6 compared with other EC types, independent of age, gender, and disease of the cell donors. In contrast, the expression of E-selectin was not affected. We conclude that the proposed isolation protocol allows the generation of a pEC bank, enabling us to study tissue-specific aspects at the level of the endothelium.

mTOR-inhibitors simultaneously inhibit proliferation and basal IL-6 synthesis of human coronary artery endothelial cells.

Divergent results regarding the immunosuppressive effects of mammalian-target-of-rapamycin-(mTOR)-inhibitors on venous endothelial cells (ECs) have highlighted the importance of an accurate EC-model. The purpose of this study was to determine mTOR-inhibitor effects at a specific site of action -- the human coronary-artery-ECs (HCAECs) -- and to compare these data with results gained from cultures of human saphenous vein ECs (HSVECs). This EC-model could enable us to gain insight into site-specific pharmacodynamics and the immunosuppressive management of transplant vasculopathy. ECs were cultivated with rising concentrations of mTOR-inhibitors in the presence/absence of tumor necrosis factor (TNF). Cell counts, DNA-synthesis, cytotoxicity and concentrations of the cytokine IL-6 as well as the chemokines IL-8 and MCP-1 were measured. Half-maximal inhibitory effects on cell growth were reached after about 30 h incubation and both cell types showed equal responses regarding cell growth and DNA-synthesis after 48 h incubation time. mTOR-inhibitors failed to suppress basal/TNF-induced secretion of IL-8 and MCP-1, but IL-6 synthesis after TNF-induction was reduced to 35%. In contrast to human saphenous vein ECs (HSVECs), mTOR-inhibitors also reduced basal IL-6-secretion of HCAECs (to 55%) and cell proliferation was simultaneously inhibited within the same concentration range. Taking everything into account, we conclude that EC-proliferation is inhibited at concentrations needed to suppress TNF-stimulated IL-6 synthesis. Furthermore, the specific suppression of basal arterial IL-6-secretion and the delayed onset of the mTOR-inhibitor effect on HCAEC-proliferation (maximum reached after about 36 h) might be of relevance for the prevention of transplant vasculopathy at its initial stage, e.g. as a component of cardioplegic solutions.

Development of a disease-specific model to evaluate endothelial dysfunction in patients with diabetes mellitus.

Diabetic patients have an increased cardiovascular risk. We propose to characterize the endothelial dysfunction in a disease-specific in vitro model. Human saphenous vein endothelial cells (HSVEC) were isolated from coronary artery bypass patients without and with non-insulin-dependent diabetes mellitus. Growth kinetics and proinflammatory responses (expression of adhesion molecules, cytokines) were documented under non-stimulating conditions. Diabetic HSVEC showed delayed growth kinetics with reduced cell densities of about 40%. During exponential growth of diabetic EC, the surface expression of adhesion molecules was increased 10-fold (p< or =0.05). However, in a monolayer the expression adapted to low levels of non-diabetic EC. In addition, diabetic EC produced significantly more soluble E-selectin, VCAM-1, IL-6 and MCP-1. Our results suggest a link between the pathologically proinflammatory basic state of diabetic EC and the endothelial dysfunction in diabetic disease. Therefore, this in vitro model could be used for investigating early dysfunction and environmental effects on pathological endothelium.