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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.

Multipotent mesenchymal stromal cells (MSC) have recently emerged as promising candidates for cell‐based immunotherapy in solid‐organ transplantation. However, optimal conditions and settings for fully harnessing MSC tolerogenic properties need to be defined. We recently reported that autologous MSC given posttransplant in kidney transplant patients was associated with transient renal insufficiency associated with intragraft recruitment of neutrophils and complement C3 deposition. Here, we moved back to a murine kidney transplant model with the aim to define the best timing of MSC infusion capable of promoting immune tolerance without negative effects on early graft function. We also investigated the mechanisms of the immunomodulatory and/or proinflammatory activities of MSC according to whether cells were given before or after transplant. Posttransplant MSC infusion in mice caused premature graft dysfunction and failed to prolong graft survival. In this setting, infused MSC localized mainly into the graft and associated with neutrophils and complement C3 deposition. By contrast, pretransplant MSC infusion induced a significant prolongation of kidney graft survival by a Treg‐dependent mechanism. MSC‐infused pretransplant localized into lymphoid organs where they promoted early expansion of Tregs. Thus, pretransplant MSC infusion may be a useful approach to fully exploit their immunomodulatory properties in kidney transplantation.

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.

We investigated the effect of hemodynamic shear forces on the expression of adhesive molecules, E-selectin, and intercellular adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVEC) exposed to laminar (8 dynes/cm2) or turbulent shear stress (8.6 dynes/cm2 average), or to a static condition. Laminar flow induced a significant time-dependent increase in the surface expression of ICAM-1, as documented by flow cytometry studies. Endothelial cell surface expression of ICAM-1 in supernatants of HUVEC exposed to laminar flow was not modified, excluding the possibility that HUVEC exposed to laminar flow synthetize factors that upregulate ICAM-1. The effect of laminar flow was specific for ICAM-1, while E-selectin expression was not modulated by the flow condition. Turbulent flow did not affect surface expression of either E-selectin or ICAM-1. To evaluate the functional significance of the laminar-flow-induced increase in ICAM-1 expression, we studied the dynamic interaction of total leukocyte suspension with HUVEC exposed to laminar flow (8 dynes/cm2 for 6 hours) in a parallel-plate flow chamber or to static condition. Leukocyte adhesion to HUVEC pre-exposed to flow was significantly enhanced, compared with HUVEC maintained in static condition (233 +/- 67 v 43 +/- 16 leukocytes/mm2, respectively), and comparable with that of interleukin-1 beta treated HUVEC. Mouse monoclonal antibody anti-ICAM-1 completely blocked flow-induced upregulation of leukocyte adhesion. Interleukin-1 beta, which upregulated E-selectin expression, caused leukocyte rolling on HUVEC that was significantly lower on flow- conditioned HUVEC and almost absent on untreated static endothelial cells. Thus, laminar flow directly and selectively upregulates ICAM-1 expression on the surface of endothelial cells and promotes leukocyte adhesion. These data are relevant to the current understanding of basic mechanisms that govern local inflammatory reactions and tissue injury.

We investigated the effects of culture medium conditioned with endothelial cells exposed to hemodynamic shear forces on modulation of mesangial cell (MC) growth. Confluent monolayers of bovine aortic endothelial cells, grown in medium containing 10% fetal calf serum, were exposed to static or to laminar flow conditions for 24 h using a cone-and-plate device. Endothelial cell-conditioned medium was used to study the growth of bovine MC by [3H]thymidine uptake. The proliferative response of MC to fresh medium (containing 10% fetal calf serum) and to culture medium from endothelial cells under static flow [66.7 +/- 34.1 vs. 73.9 +/- 30.0 counts/min (cpm) x 10(-3)] was comparable. In contrast, medium conditioned with endothelial cells exposed to laminar shear stress of 8 dyn/cm2 almost completely abolished MC proliferation (5.8 +/- 6.9 cpm x 10(-3), P < 0.01). To establish whether this effect is due to endothelial cell production of a substance that inhibits MC proliferation or simply to metabolization of serum growth factors in the culture medium, we performed shear stress experiments using serum free medium and we added 10% fetal calf serum after shear exposure just before the proliferation assay. In this condition a significant antiproliferative effect of endothelial cell supernatant under laminar flow was obtained (27.7 +/- 23.4 vs. 68.8 +/- 45.8 cpm x 10(-3), laminar vs. static, P < 0.05), suggesting that endothelial cells under shear stress effectively produce a factor that inhibits MC proliferation. These results would suggest that local glomerular capillary blood flow could play a role in the regulation of MC mitogenesis.

Platelet-activating factor (PAF) has been suggested recently to play an important role in immune glomerulonephritis, favoring the formation of immune deposits in glomeruli and contributing to the local inflammatory reaction. Here we sought to investigate whether urinary PAF excretion was modified in New Zealand Black x New Zealand White mice a model of genetically determined immune complex disease which mimics systemic lupus in humans and whether changes in PAF urinary excretion values correlated with the extent of proteinuria. To clarify the possible "in vivo" relevance of these findings we evaluated whether PAF receptor antagonist has any influence on the evolution of renal disease and survival of these mice. Our results showed that: 1) in lupus mice urinary PAF excretion increased progressively with age in New Zealand Black x White; 2) the increase in PAF excretion correlated with the severity of proteinuria; and 3) the chronic administration of a PAF receptor antagonist [L-659,989 [(+/- )-trans-2-(3-methoxy-5-methylsulfonyl-4-propoxyphenyl)-5- (3,4,5-trimethoxyphenyl)tetrahydrofuran]] starting from 26 weeks of age significantly delayed the onset of proteinuria and prolonged survival.