A novel strategy to enhance mesenchymal stem cell migration capacity and promote tissue repair in an injury specific fashion.

Mesenchymal stem cells (MSCs) of bone marrow origin appear to be an attractive candidate for cell-based therapies. However, the major barrier to the effective implementation of MSC-based therapies is the lack of specific homing of exogenously infused cells and overall the inability to drive them to the diseased or damaged tissue. In order to circumvent these limitations, we developed a preconditioning strategy to optimize MSC migration efficiency and potentiate their beneficial effect at the site of injury. Initially, we screened different molecules by using an in vitro injury-migration setting, and subsequently, we evaluated the effectiveness of the different strategies in mice with acute kidney injury (AKI). Our results showed that preconditioning of MSCs with IGF-1 before infusion improved cell migration capacity and restored normal renal function after AKI. The present study demonstrates that promoting migration of MSCs could increase their therapeutic potential and indicates a new therapeutic paradigm for organ repair.

The response of endothelial cells to fluid shear stress using a co-culture model of the arterial wall.

An endothelial cell (EC) smooth muscle cell (SMC) co-culture model of the arterial wall was used to study the effect of fluid shear stress on EC behavior. This model, in addition to being a more realistic tissue analogue, is a valuable research tool for studying the effects of mechanical stimulation upon the behavior of both SMCs and ECs. In the present study, a 10% cyclic strain was used to alter the characteristics of an SMC-seeded collagen gel. This form of strain preconditioning resulted in a rearrangement of the vessel wall that yielded circumferentially oriented cells and collagen fibrils. The preconditioned collagen gel was subsequently seeded with ECs and exposed to fluid-induced shear stress (10 dynes/cm2) for 48 hr. In the absence of flow, ECs seeded on slab constructs were oriented with the underlying collagen fibrils. Sheared constructs exhibited ECs oriented in the flow direction. Shear stress also affected EC proliferation, reducing the total number of dividing ECs by as much as 48 percent compared to unsheared constructs. The shear-induced reduction in proliferation was further enhanced when constructs were first strain-preconditioned (64% reduction). Moreover, conditioned media from shear stress experiments inhibited proliferation of ECs seeded on tissue culture plastic. These results suggest that EC response to fluid shear stress in a collagen co-culture model is influenced by the underlying substrate, and one that in this study is modified by strain preconditioning.

Verotoxin-1-induced up-regulation of adhesive molecules renders microvascular endothelial cells thrombogenic at high shear stress.

Verotoxin-1 (VT-1)-producing Escherichia coli is the causative agent of postdiarrheal hemolytic uremic syndrome (D+HUS) of children, which leads to renal and other organ microvascular thrombosis. Why thrombi form only on arterioles and capillaries is not known. This study investigated whether VT-1 directly affected endothelial antithrombogenic properties promoting platelet deposition and thrombus formation on human microvascular endothelial cell line (HMEC-1) under high shear stress. Human umbilical vein endothelial cells (HUVECs) were used for comparison as a large-vessel endothelium. HMEC-1 and HUVECs were pre-exposed for 24 hours to increasing concentrations of VT-1 (2-50 pM) and then perfused at 60 dynes/cm(2) with heparinized human blood prelabeled with mepacrine. Results showed that VT-1 significantly increased platelet adhesion and thrombus formation on HMEC-1 in comparison with unstimulated control cells. An increase in thrombus formation was also observed on HUVECs exposed to VT-1, but to a remarkably lower extent. The greater sensitivity of HMEC-1 to the toxin in comparison with HUVECs was at least in part due to a higher expression of VT-1 receptor (20-fold more) as documented by FACS analysis. The HMEC-1 line had a comparable susceptibility to the thrombogenic effect of VT-1 as primary human microvascular cells of the same dermal origin (HDMECs). The adhesive molecules involved in VT-induced thrombus formation were also studied. Blocking the binding of von Willebrand factor to platelet glycoprotein Ib by aurintricarboxylic acid (ATA) or inhibition of platelet alpha(IIb)beta(3)-integrin by chimeric 7E3 Fab resulted in a significant reduction of VT-1-induced thrombus formation, suggesting the involvement of von Willebrand factor-platelet interaction at high shear stress in this phenomenon. Functional blockade of endothelial beta(3)-integrin subunit, vitronectin receptor, P-selectin, and PECAM-1 with specific antibodies was associated with a significant decrease of the endothelial area covered by thrombi. Confocal microscopy studies revealed that VT-1 increased the expression of vitronectin receptor and P-selectin and redistributed PECAM-1 away from the cell-cell border of HMEC-1, as well as of HDMECs, thus indicating that the above endothelial adhesion molecules are directly involved and possibly determine the effect of VT-1 on enhancing platelet adhesion and thrombus formation in microvascular endothelium. These results might help to explain why thrombi in HUS localize in microvessels rather than in larger ones and provide insights on the molecular events involved in the process of microvascular thrombosis associated with D+HUS.

Xenogeneic serum promotes leukocyte-endothelium interaction under flow through two temporally distinct pathways: role of complement and nuclear factor-kappaB.

Endothelial cell activation and mononuclear cell infiltration are consistent features of discordant xenograft rejection. This study evaluated whether xenogeneic serum--as a source of xenoreactive natural antibodies and complement--induced endothelial activation with consequent leukocyte adhesion and transmigration under flow conditions. Porcine aortic endothelial cells (PAEC) were incubated for 30 min, 1 h 30 min, or 5 h with 10% human serum or 10% porcine serum and then perfused with human leukocytes in a parallel plate flow chamber under flow (1.5 dynes/cm2). Adherent and transmigrated cells were counted by digital image analysis. Results showed that human serum significantly (P < 0.01) increased over time the number of adherent leukocytes compared with porcine serum. Stimulation of PAEC with human serum also promoted a progressive increase in leukocyte transmigration that reached statistical significance (P < 0.01) at 1 h 30 min and at 5 h compared with porcine serum. Studying the role of complement in leukocyte-endothelium interaction in xenogeneic conditions, a marked complement C3 deposition on PAEC exposed to human serum was shown by immunofluorescence, whereas cells incubated with porcine serum were negative. Next, it was documented that human serum decomplemented by heating and C3-deficient human serum failed to promote both leukocyte adhesion and transmigration, results that were comparable to porcine serum. To elucidate the intracellular mediators involved in endothelial cell activation by xenogeneic serum, this study focused on transcriptional factor nuclear factor-kappaB (NF-kappaB), a central regulator for the induction of different genes, including adhesive molecules and chemoattractants. Positive nuclear staining of NF-kappaB (p65 subunit) found by confocal fluorescence microscopy of PAEC exposed to human serum was taken to reflect NF-kappaB activation. NF-kappaB was instead strictly localized in the cell cytoplasm in PAEC incubated with the homologous serum. Heat-inactivated human serum failed to activate NF-kappaB. Electrophoretic mobility shift assay of nuclear extracts from PAEC exposed to human serum revealed an intense NF-kappaB activation that was inhibited by the NF-kappaB inhibitor pyrrolidinedithiocarbamate. The NF-kappaB inhibitors pyrrolidinedithiocarbamate and tosyl-phe-chloromethylketone did not affect the number of adherent and transmigrated leukocytes in PAEC exposed to human serum for 30 min and 1 h 30 min. Both inhibitors instead significantly reduced leukocyte adhesion and transmigration induced by human serum at 5 h. Confocal fluorescence microscopy studies showed that human serum induced an increase in the expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1. Functional blocking of these adhesive molecules with the corresponding antibodies significantly inhibited xenogeneic serum-induced leukocyte adhesion. These data suggest that leukocyte adhesion and transmigration are directly dependent on complement deposited on PAEC in the early phase of cell activation (30 min and 1 h 30 min) induced by xenogeneic serum, whereas leukocyte adhesive events observed after 5 h of incubation of endothelial cells with xenogeneic serum are possibly regulated by transcription of NF-kappaB-dependent genes. The finding that xenogeneic serum promotes leukocyte-endothelial interaction depending on NF-kappaB activation might be relevant for designing future therapeutic strategies intended to prolong xenograft survival.

Xenogeneic human serum promotes leukocyte adhesion to porcine endothelium under flow conditions, possibly through the activation of the transcription factor NF-kappa B.

Endothelial cell activation and leukocyte infiltration are a consistent feature of discordant xenograft rejection. Here we evaluated whether xenogeneic serum, as a source of xenoreactive natural antibodies and complement, induced endothelial cell activation with consequent leukocyte adhesion under flow conditions. Porcine aortic endothelial cells (PAEC) were incubated for 1 hr 30 min or 5 hr with 10% homologous porcine serum (control) or 10% xenogeneic human serum and then perfused with total human leukocytes in a parallel plate flow chamber under laminar flow (1.5 dynes/cm2). Adherent cells were counted by digital image analysis. Xenogeneic human serum significantly (P < 0.01) increased the number of adherent leukocytes as compared with porcine serum. A similar adhesive response was elicited by TNF alpha (100 U/ml), one of the most potent inducers of endothelial cell adhesive properties, here used as positive control. In order to elucidate possible mechanisms underlying endothelial cell activation by xenogeneic serum, we focussed on transcription factor NF-kappa B, a central regulator for the induction of different genes, including adhesive molecules and chemoattractants. By confocal fluorescence microscopy, we observed a positive staining for NF-kappa B (p65 subunit) in the nuclei of PAEC exposed for 1 hr 30 min to human serum, which indicated NF-kappa B activation in this setting. At variance, in PAEC incubated with the homologous serum, NF-kappa B was strictly localized in the cell cytoplasm. Treatment of PAEC exposed to xenogeneic serum with the NF-kappa B inhibitors pyrrolidinedithiocarbamate (PDTC, 25 microM) and tosyl-phechloromethylketone (TPCK, 25 microM) significantly (P < 0.01) reduced leukocyte adhesion in respect to PAEC treated with human serum alone. Findings that xenogeneic serum promotes leukocyte-endothelium interaction possibly through NF-kappa B activation might be relevant for designing future therapeutic strategies aimed at prolonging xenograft survival.

Effect of acetate, bicarbonate dialysis, and acetate-free biofiltration on nitric oxide synthesis: implications for dialysis hypotension.

The effect of acetate dialysis (AD), bicarbonate dialysis (BD), and acetate-free biofiltration (AFB) on nitric oxide (NO) synthesis and the implications for dialysis hypotension was studied. The finding that uremic plasma is a potent inducer of NO synthesis by endothelial cells in vitro suggested that the cardiovascular instability of dialysis patients might result from excessive NO formation. Cardiovascular instability is more frequent in patients undergoing AD than BD. To see whether these differences were attributable to NO, we studied the NO synthetic pathway ex vivo in patients undergoing different dialysis procedures. Five patients were treated, in a random order, with AD, BD, and AFB, a technique using a buffer-free dialysate and postdilution of a sterile bicarbonate solution. Each type of dialysis was used for 1 week, comprising three dialysis sessions. A polyacrylonitrile dialyzer was used for all three methods. Before and after the third dialysis, plasma was collected, added to [3H]L-arginine, and incubated with human umbilical vein endothelial cells (HUVECs) for 24 hours. NO synthesis was evaluated as [3H]L-citrulline formation. Plasma concentrations of interleukin-1beta (IL-1beta), a potent inducer of inducible NO synthase (iNOS) in endothelial cells, were also measured. Plasma collected from patients after AD stimulated endothelial NO synthesis more than plasma from the same patients before the dialysis session (pre-AD, 0.173+/-0.028 nmol/10(5) cells v post-AD, 0.280+/-0.093 nmol/10(5) cells; P < 0.05). A slight, although not significant, increase was also observed when HUVECs were incubated with plasma drawn after BD (pre-BD, 0.151+/-0.014 nmol/10(5) cells; post-BD, 0.230+/-0.055 nmol/10(5) cells). AFB did not aggravate the stimulatory effect of uremic plasma on endothelial NO synthesis (pre-AFB, 0.184+/-0.038 nmol/10(5) cells; post-AFB, 0.189+/-0.040 nmol/10(5) cells). Plasma IL-1beta was greater (P < 0.01) after AD than after BD and AFB (post-AD, 0.234+/-0.028 pg/mL; post-BD, 0.124+/-0.019 pg/mL; post-AFB, 0.120+/-0.013 pg/mL). With AD, there was a greater intradialytic decrease in systolic blood pressure than with BD or AFB. Weight and blood volume loss and sodium balance were similar in AD, BD, and AFB. These data were consistent with the possibility that NO and cytokines, released in excessive amounts during AD, may contribute to hemodynamic instability.

Leukocyte-endothelial interaction is augmented by high glucose concentrations and hyperglycemia in a NF-kB-dependent fashion.

We addressed the role of hyperglycemia in leukocyte-endothelium interaction under flow conditions by exposing human umbilical vein endothelial cells for 24 h to normal (5 mM), high concentration of glucose (30 mM), advanced glycosylation end product-albumin (100 microg/ml), or hyperglycemic (174-316 mg/dl) sera from patients with diabetes and abnormal hemoglobin A1c (8.1+/-1.4%). At the end of incubation endothelial cells were perfused with total leukocyte suspension in a parallel plate flow chamber under laminar flow (1.5 dyn/cm2). Rolling and adherent cells were evaluated by digital image processing. Results showed that 30 mM glucose significantly (P < 0. 01) increased the number of adherent leukocytes to endothelial cells in respect to control (5 mM glucose; 151+/-19 versus 33+/-8 cells/mm2). A similar response was induced by endothelial stimulation with IL-1beta, here used as positive control (195+/-20 cells/mm2). The number of rolling cells on endothelial surface was not affected by high glucose level. Stable adhesion of leukocytes to glucose-treated as well as to IL-1beta-stimulated endothelial cells was preceded by short interaction of leukocytes with the endothelial surface. The distance travelled by leukocytes before arrest on 30 mM glucose, or on IL-1beta-treated endothelial cells, was significantly (P < 0.01) higher than that observed for leukocytes adhering on control endothelium (30 mM glucose: 76.7+/-3.5; IL1beta: 69.7+/-4 versus 5 mM glucose: 21.5+/-5 microm). Functional blocking of E-selectin, intercellular cell adhesion molecule-1, and vascular cell adhesion molecule-1 on endothelial cells with the corresponding mouse mAb significantly inhibited glucose-induced increase in leukocyte adhesion (67+/-16, 83+/-12, 62+/-8 versus 144+/-21 cells/ mm2). Confocal fluorescence microscopy studies showed that 30 mM glucose induced an increase in endothelial surface expression of E-selectin, intercellular cell adhesion molecule-1, and vascular cell adhesion molecule-1. Electrophoretic mobility shift assay of nuclear extracts of human umbilical vein endothelial cells (HUVEC) exposed for 1 h to 30 mM glucose revealed an intense NF-kB activation. Treatment of HUVEC exposed to high glucose with the NF-kB inhibitors pyrrolidinedithiocarbamate (100 microM) and tosyl-phe-chloromethylketone (25 microM) significantly reduced (P < 0.05) leukocyte adhesion in respect to HUVEC treated with glucose alone. A significant (P < 0.01) inhibitory effect on glucose-induced leukocyte adhesion was observed after blocking protein kinase C activity with staurosporine (5 nM). When HUVEC were treated with specific antisense oligodesoxynucleotides against PKCalpha and PKCepsilon isoforms before the addition of 30 mM glucose, a significant (P < 0.05) reduction in the adhesion was also seen. Advanced glycosylation end product-albumin significantly increased the number of adhering leukocytes in respect to native albumin used as control (110+/-16 versus 66+/-7, P < 0.01). Sera from diabetic patients significantly (P < 0.01) enhanced leukocyte adhesion as compared with controls, despite normal levels of IL-1beta and TNFalpha in these sera. These data indicate that high glucose concentration and hyperglycemia promote leukocyte adhesion to the endothelium through upregulation of cell surface expression of adhesive proteins, possibly depending on NF-kB activation.

Cyclosporine enhances leukocyte adhesion to vascular endothelium under physiologic flow conditions.

We investigated the effect of cyclosporine (CyA) on leukocyte adhesion to endothelium under flow conditions. Confluent human umbilical vein endothelial cells (HUVECs) were incubated for 24 hours with CyA (1, 5, and 10 micromol/L) and then exposed to a total human leukocyte suspension in a parallel plate flow chamber under laminar flow (1.5 dynes/cm2. Human umbilical vein endothelial cells stimulated with interleukin-1beta (20 U/mL) were used as a positive control. Adherent cells were measured by digital image analysis. Results showed that CyA dose-dependently increased the number of leukocytes adhering to HUVECs compared with control cells. Leukocyte adhesion markedly increased on HUVECs incubated with interleukin-lbeta, one of the most potent inducers of endothelial cell adhesiveness. Exposure of endothelial cells to CyA did not affect the number of rolling leukocytes, which was similar to control values. To examine the role of adhesion molecules in CyA-induced leukocyte adhesion, HUVECs were incubated with monoclonal antibodies against intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and E-selectin before adhesion assay. Functional blocking of ICAM-1, VCAM-1, and E-selectin on endothelial cells significantly inhibited CyA (10 micromol/L)-induced leukocyte adhesion. Confocal fluorescence microscopy studies showed that CyA induced an increase in the endothelial surface expression of ICAM-1, VCAM-1, and E-selectin. Pretreatment of leukocytes with the platelet activating factor receptor antagonist L659,989 significantly reduced the number of leukocytes adhering to CyA-treated HUVECs. We suggest that CyA enhances leukocyte adhesion to endothelium by upregulating adhesive proteins on endothelial surface membrane. Blocking leukocyte receptor for platelet-activating factor partially prevents adhesion, suggesting a role for endothelial cell-associated platelet-activating factor in the interaction between leukocytes and CyA-treated endothelium.

Verotoxin-1 promotes leukocyte adhesion to cultured endothelial cells under physiologic flow conditions.

Hemolytic uremic syndrome (HUS), which is the most common cause of acute renal failure in infants and small children, is caused by verotoxin (VT)-producing Escherichia coli infection. Endothelial injury determines microvascular thrombosis and evidence is available from recent studies that suggests that leukocyte activation participates in endothelial damage. We studied here the effect of VT-1 on leukocyte adhesion to vascular endothelium under physiologic flow conditions. Human umbilical vein endothelial cells (HUVECs) were incubated for 24 hours with VT-1 (0.1, 1, and 10 pmol/L) and then exposed to a total leukocyte suspension in a parallel plate flow chamber under laminar flow conditions (1.5 dynes/cm2). Adherent cells were counted by digital image processing. Results showed that VT-1 dose-dependently increased the number of adhering leukocytes to HUVECs as compared with unstimulated cells. The adhesive response elicited by VT-1 was comparable to that of interleukin-1 beta (IL-1 beta), one of the most potent inducers of endothelial cell adhesiveness. Exposure of HUVECs to VT-1 did not affect the number of rolling leukocytes, which was similar to that of control values. To examine the role of adhesion molecules in VT-1-induced leukocyte adhesion, HUVECs were incubated with mouse monoclonal antibodies against E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) before adhesion assay. Functional blocking of E-selectin, ICAM-1, and VCAM-1 on endothelial cells significantly inhibited VT-1-induced increase in leukocyte adhesion. In some experiments, before VT-1 incubation, HUVECs were pretreated for 24 hours with tumor necrosis factor alpha (TNF alpha; 100 U/mL), which is known to increase VT receptor expression on HUVECs. The number of adhering leukocytes on HUVECs exposed to TNF alpha and VT-1 significantly increased as compared with HUVECs incubated with VT-1 and TNF alpha alone. These results suggest that VT-1 modulates leukocyte-endothelium interaction, thus increasing leukocyte adhesion and upregulating adhesive proteins on endothelial surface membrane.