Pascal Rowart

Mesenchymal Stromal Cell Therapy in Ischemia/Reperfusion Injury.

Ischemia/reperfusion injury (IRI) represents a worldwide public health issue of increasing incidence. IRI may virtually affect all organs and tissues and is associated with significant morbidity and mortality. Particularly, the duration of blood supply deprivation has been recognized as a critical factor in stroke, hemorrhagic shock, or myocardial infarction, as well as in solid organ transplantation (SOT). Pathophysiologically, IRI causes multiple cellular and tissular metabolic and architectural changes. Furthermore, the reperfusion of ischemic tissues induces both local and systemic inflammation. In the particular field of SOT, IRI is an unavoidable event, which conditions both short- and long-term outcomes of graft function and survival. Clinically, the treatment of patients with IRI mostly relies on supportive maneuvers since no specific target-oriented therapy has been validated thus far. In the present review, we summarize the current literature on mesenchymal stromal cells (MSC) and their potential use as cell therapy in IRI. MSC have demonstrated immunomodulatory, anti-inflammatory, and tissue repair properties in rodent studies and in preliminary clinical trials, which may open novel avenues in the management of IRI and SOT.

Mesenchymal Stromal Cells Accelerate Epithelial Tight Junction Assembly via the AMP-Activated Protein Kinase Pathway, Independently of Liver Kinase B1

Background Mesenchymal stromal cells (MSC) are fibroblast-like multipotent cells capable of tissue-repair properties. Given the essentiality of tight junctions (TJ) in epithelial integrity, we hypothesized that MSC modulate TJ formation, via the AMP-activated kinase (AMPK) pathway. Liver kinase-β1 (LKB1) and Ca2+-calmodulin-dependent protein kinase kinase (CaMKK) represent the main kinases that activate AMPK. Methods The in vitro Ca2+ switch from 5 μM to 1.8 mM was performed using epithelial Madin-Darby canine kidney (MDCK) cells cultured alone or cocultured with rat bone marrow-derived MSC or preexposed to MSC-conditioned medium. TJ assembly was measured by assessing ZO-1 relocation to cell-cell contacts. Experiments were conducted using MDCK stably expressing short-hairpin-RNA (shRNA) against LKB1 or luciferase (LUC, as controls). Compound STO-609 (50 μM) was used as CaMKK inhibitor. Results Following Ca2+ switch, ZO-1 relocation and phosphorylation/activation of AMPK were significantly higher in MDCK/MSC compared to MDCK. No difference in AMPK phosphorylation was observed between LKB1-shRNA and Luc-shRNA MDCK following Ca2+ switch. Conversely, incubation with STO-609 prior to Ca2+ switch prevented AMPK phosphorylation and ZO-1 relocation. MSC-conditioned medium slightly but significantly increased AMPK activation and accelerated TJ-associated distribution of ZO-1 post Ca2+ switch in comparison to regular medium. Conclusions MSC modulate the assembly of epithelial TJ, via the CaMKK/AMPK pathway independently of LKB1.

Administration of mesenchymal stromal cells before renal ischemia/reperfusion attenuates kidney injury and may modulate renal lipid metabolism in rats.

Mesenchymal stromal cells (MSC) have been demonstrated to attenuate renal ischemia/reperfusion (I/R) damage in rodent models. The mechanisms of such nephro-protection remain largely unknown. Furthermore, the optimal timing of MSC administration has been poorly investigated. Here, we compare the impact of MSC injection 7 days before (MSCD − 7) versus 1 day after (MSCD + 1) renal I/R in rats. Control groups received equivalent volumes of saline at similar time-points (SD − 7 and SD + 1). Right nephrectomy was performed, and left renal ischemia lasted 45 min. After 48-hour reperfusion, we observed significantly improved renal function parameters, reduced apoptotic index and neutrophil/macrophage infiltration in kidney parenchyma, and lower expression of tubular damage markers and pro-inflammatory cytokines in MSCD − 7 in comparison to MSCD + 1 and saline control groups. Next, comparative high-throughput RNA sequencing of MSCD − 7 vs. SD − 7 non-ischemic right kidneys highlighted significant down-regulation of fatty acid biosynthesis and up-regulation of PPAR-α pathway. Such a preferential regulation towards lipid catabolism was associated with decreased levels of lipid peroxidation products, i.e. malondialdehyde and 4-hydroxy-2-nonenal, in MSCD − 7 versus SD − 7 ischemic kidneys. Our findings suggest that MSC pretreatment may exert protective effects against renal I/R by modulating lipid metabolism in rats.

Implications of AMPK in the Formation of Epithelial Tight Junctions

Tight junctions (TJ) play an essential role in the epithelial barrier. By definition, TJ are located at the demarcation between the apical and baso-lateral domains of the plasma membrane in epithelial cells. TJ fulfill two major roles: (i) TJ prevent the mixing of membrane components; and (ii) TJ regulate the selective paracellular permeability. Disruption of TJ is regarded as one of the earliest hallmarks of epithelial injury, leading to the loss of cell polarity and tissue disorganization. Many factors have been identified as modulators of TJ assembly/disassembly. More specifically, in addition to its role as an energy sensor, adenosine monophosphate-activated protein kinase (AMPK) participates in TJ regulation. AMPK is a ubiquitous serine/threonine kinase composed of a catalytic α-subunit complexed with regulatory β-and γ-subunits. AMPK activation promotes the early stages of epithelial TJ assembly. AMPK phosphorylates the adherens junction protein afadin and regulates its interaction with the TJ-associated protein zonula occludens (ZO)-1, thereby facilitating ZO-1 distribution to the plasma membrane. In the present review, we detail the signaling pathways up-and down-stream of AMPK activation at the time of Ca2+-induced TJ assembly.

What we need to know about lipid-associated injury in case of renal ischemia/reperfusion

Renal segmental metabolism is reflected by the complex distribution of the main energy pathways along the nephron, with fatty acid oxidation preferentially used in the cortex area. Ischemia/reperfusion injury (IRI) is due to the restriction of renal blood flow, rapidly leading to a metabolic switch toward anaerobic conditions. Subsequent unbalance between energy demand and oxygen/nutrient delivery compromises kidney cell functions, resulting in a complex inflammatory cascade including the production of reactive oxygen species (ROS). Renal IRI especially involves lipid accumulation. Lipid peroxidation is one of the major events of ROS-associated tissue injury. Here, we briefly review the current knowledge of renal cell lipid metabolism in normal and ischemic conditions. Next, we focus on renal lipid-associated injury, with emphasis on its mechanisms and consequences during the course of IRI. Finally, we discuss preclinical observations aiming at preventing and/or attenuating lipid-associated IRI.