Joseph Zullo

Curtailing Endothelial TGF-β Signaling Is Sufficient to Reduce Endothelial-Mesenchymal Transition and Fibrosis in CKD

Excessive TGF-β signaling in epithelial cells, pericytes, or fibroblasts has been implicated in CKD. This list has recently been joined by endothelial cells (ECs) undergoing mesenchymal transition. Although several studies focused on the effects of ablating epithelial or fibroblast TGF-β signaling on development of fibrosis, there is a lack of information on ablating TGF-β signaling in the endothelium because this ablation causes embryonic lethality. We generated endothelium-specific heterozygous TGF-β receptor knockout (TβRII(endo+/-)) mice to explore whether curtailed TGF-β signaling significantly modifies nephrosclerosis. These mice developed normally, but showed enhanced angiogenic potential compared with TβRII(endo+/+) mice under basal conditions. After induction of folic acid nephropathy or unilateral ureteral obstruction, TβRII(endo+/-) mice exhibited less tubulointerstitial fibrosis, enhanced preservation of renal microvasculature, improvement in renal blood flow, and less tissue hypoxia than TβRII(endo+/+) counterparts. In addition, partial deletion of TβRII in the endothelium reduced endothelial-to-mesenchymal transition (EndoMT). TGF-β-induced canonical Smad2 signaling was reduced in TβRII(+/-) ECs; however, activin receptor-like kinase 1 (ALK1)-mediated Smad1/5 phosphorylation in TβRII(+/-) ECs remained unaffected. Furthermore, the S-endoglin/L-endoglin mRNA expression ratio was significantly lower in TβRII(+/-) ECs compared with TβRII(+/+) ECs. These observations support the hypothesis that EndoMT contributes to renal fibrosis and curtailing endothelial TGF-β signals favors Smad1/5 proangiogenic programs and dictates increased angiogenic responses. Our data implicate endothelial TGF-β signaling and EndoMT in regulating angiogenic and fibrotic responses to injury.

Endothelial sirtuin 1 inactivation enhances capillary rarefaction and fibrosis following kidney injury through Notch activation.

Peritubular capillary (PTC) rarefaction along with tissue fibrosis is a hallmark of chronic kidney disease (CKD). However, molecular mechanisms of PTC loss have been poorly understood. Previous studies have demonstrated that functional loss of endothelial sirtuin 1 (SIRT1) impairs angiogenesis during development and tissue damage. Here, we found that endothelial SIRT1 dysfunction causes activation of endothelial Notch1 signaling, which leads to PTC rarefaction and fibrosis following kidney injury. In mice lacking functional SIRT1 in the endothelium (Sirt1 mutant), kidney injury enhanced apoptosis and senescence of PTC endothelial cells with impaired endothelial proliferation and expanded myofibroblast population and collagen deposition. Compared to wild-type kidneys, Sirt1 mutant kidneys up-regulated expression of Delta-like 4 (DLL4, a potent Notch1 ligand), Hey1 and Hes1 (Notch target genes), and Notch intracellular domain-1 (NICD1, active form of Notch1) in microvascular endothelial cells (MVECs) post-injury. Sirt1 mutant primary kidney MVECs reduced motility and vascular assembly and enhanced senescence compared to wild-type kidney MVECs. This difference in the phenotype was negated with Notch inhibition. Concurrent stimulation of DLL4 and transforming growth factor (TGF)-β1 increased trans-differentiation of primary kidney pericytes into myofibroblast more than TGF-β1 treatment alone. Collectively, these results indicate that endothelial SIRT1 counteracts PTC rarefaction by repression of Notch1 signaling and antagonizes fibrosis via suppression of endothelial DLL4 expression.

Therapeutic Restoration of Endothelial Glycocalyx in Sepsis.

Endothelial glycocalyx (EG) is disintegrated during sepsis. We have previously shown that this occurs very early in the course of sepsis and its prevention improves the survival of mice with sepsis. Here, we sought to investigate the possibility of pharmacologically accelerating the restoration of disintegrated EG in sepsis. We used a soilage injection model to induce polymicrobial sepsis in C57/BL6 mice and measured total body EG. En face aortic preparations were used for staining of markers of EG and atomic force microscopy was used to measure EG in vitro. In vitro studies were conducted in cultured endothelial cells either exposed to a lipopolysaccharide or enzymatically denuded of EG. Sulodexide (SDX), a heparin sulfate-like compound resistant to degradation by heparanase, accelerated EG regeneration in vitro and in vivo. The total volume of EG was drastically reduced in septic mice. Administration of SDX produced a dramatic acceleration of EG restoration. This effect, unrelated to any SDX-induced differences in microbial burden, was associated with better control of vascular permeability. Notably, SDX demonstrated not only a remarkable capacity for EG regeneration in vitro and in vivo but was also associated with improved animal survival, even when instituted 2 hours after induction of severe sepsis. In conclusion, 1) EG is disintegrated in sepsis, the event which contributes to high animal mortality; 2) pharmacologic acceleration of EG restoration can be achieved using SDX; and 3) SDX reduces vascular permeability, which is elevated in septic mice, and improves animal survival.

Exocytosis of Endothelial Lysosome-Related Organelles Hair-Triggers a Patchy Loss of Glycocalyx at the Onset of Sepsis.

Sepsis is a systemic inflammatory syndrome induced by bacterial infection that can lead to multiorgan failure. Endothelial surface glycocalyx (ESG) decorating the inner wall of blood vessels is a regulator of multiple vascular functions. Here, we tested a hypothesis that patchy degradation of ESG occurs early in sepsis and is a result of exocytosis of lysosome-related organelles. Time-lapse video microscopy revealed that exocytosis of Weibel-Palade bodies and secretory lysosomes occurred a few minutes after application of lipopolysaccharides to endothelial cells. Two therapeutic maneuvers, a nitric oxide intermediate, NG-hydroxy-l-arginine, and culture media conditioned by endothelial progenitor cells reduced the motility of lysosome-related organelles. Confocal and stochastic optical reconstruction microscopy confirmed the patchy loss of ESG simultaneously with the exocytosis of lysosome-related organelles and Weibel-Palade bodies in cultured endothelial cells and mouse aorta. The loss of ESG was blunted by pretreatment with NG-hydroxy-l-arginine or culture media conditioned by endothelial progenitor cells. Moreover, these treatments resulted in a significant reduction in deaths of septic mice. Our data support the hypothesis assigning to stress-induced exocytosis of these organelles the role of a hair-trigger for local degradation of ESG that initiates leukocyte infiltration, increase in vascular permeability, and partially accounts for the later rates of morbidity and mortality.

The Secretome of Hydrogel-Coembedded Endothelial Progenitor Cells and Mesenchymal Stem Cells Instructs Macrophage Polarization in Endotoxemia

We previously reported the delivery of endothelial progenitor cells (EPCs) embedded in hyaluronic acid‐based (HA)‐hydrogels protects renal function during acute kidney injury (AKI) and promotes angiogenesis. We attempted to further ameliorate renal dysfunction by coembedding EPCs with renal mesenchymal stem cells (MSCs), while examining their paracrine influence on cytokine/chemokine release and proinflammatory macrophages. A live/dead assay determined whether EPC‐MSC coculturing improved viability during lipopolysaccharide (LPS) treatment, and HA‐hydrogel‐embedded delivery of cells to LPS‐induced AKI mice was assessed for effects on mean arterial pressure (MAP), renal blood flow (RBF), circulating cytokines/chemokines, serum creatinine, proteinuria, and angiogenesis (femoral ligation). Cytokine/chemokine release from embedded stem cells was examined, including effects on macrophage polarization and release of proinflammatory molecules. EPC‐MSC coculturing improved stem cell viability during LPS exposure, an effect augmented by MSC hypoxic preconditioning. The delivery of coembedded EPCs with hypoxic preconditioned MSCs to AKI mice demonstrated additive improvement (compared with EPC delivery alone) in medullary RBF and proteinuria, with comparable effects on serum creatinine, MAP, and angiogenesis. Exposure of proinflammatory M1 macrophages to EPC‐MSC conditioned medium changed their polarization to anti‐inflammatory M2. Incubation of coembedded EPCs‐MSCs with macrophages altered their release of cytokines/chemokines, including enhanced release of anti‐inflammatory interleukin (IL)‐4 and IL‐10. EPC‐MSC delivery to endotoxemic mice elevated the levels of circulating M2 macrophages and reduced the circulating cytokines/chemokines. In conclusion, coembedding EPCs‐MSCs improved their resistance to stress, impelled macrophage polarization from M1 to M2 while altering their cytokine/chemokines release, reduced circulating cytokines/chemokines, and improved renal and vascular function when MSCs were hypoxically preconditioned.

Low birth weight is associated with impaired murine kidney development and function

BackgroundLow birth weight (LBW) neonates have impaired kidney development that leaves them susceptible to kidney disease and hypertension during adulthood. The study here identifies events that blunt nephrogenesis and kidney development in the murine LBW neonate.MethodsWe examined survival, kidney development, GFR, gene expression, and cyto-/chemokines in the LBW offspring of malnourished (caloric and protein-restricted) pregnant mice.ResultsMalnourished pregnant mothers gave birth to LBW neonates that had 40% reduced body weight and 54% decreased survival. Renal blood perfusion was reduced by 37%, whereas kidney volume and GFR were diminished in the LBW neonate. During gestation, the LBW neonatal kidney had 2.2-fold increased apoptosis, 76% decreased SIX2+ progenitor cells, downregulation of mesenchymal-to-epithelial signaling factors Wnt9b and Fgf8, 64% less renal vesicle formation, and 32% fewer nephrons than controls. At birth, increased plasma levels of IL-1β, IL-6, IL-12(p70), and granulocyte-macrophage colony-stimulating factor in the LBW neonate reduced SIX2+ progenitor cells.ConclusionIncreased pro-inflammatory cytokines in the LBW neonate decrease SIX2+ stem cells in the developing kidney. Reduced renal stem cells (along with the decreased mesenchymal-to-epithelial signaling) blunt renal vesicle generation, nephron formation, and kidney development. Subsequently, the mouse LBW neonate has reduced glomeruli volume, renal perfusion, and GFR.

The cell secretome, a mediator of cell-to-cell communication.

We are witnessing the emergence of a novel type of biological regulation, namely, the communication between cells via their secreted substances, the secretome. This brief overview is based on the available published data and our own experience. We discuss three vignettes illustrating the importance of communication via the secretome: (1) the secretome of stem cells and its effects in sepsis and systemic inflammatory response; (2) the profibrotic secretomes partially responsible for development of fibrotic complications; and (3) the contribution of senescence-associated secretory products to the propagation of the senescence phenotype. Considering the richness of secretomes of different cells under diverse conditions, it becomes imperative to gain insights into their individual components in an attempt to harness cell secretomes for therapeutic purposes.

Endothelial glycocalyx—the battleground for complications of sepsis and kidney injury

After briefly discussing endothelial glycocalyx and its role in vascular physiology and renal disease, this overview focuses on its degradation very early in the course of microbial sepsis. We describe our recently proposed mechanism for glycocalyx degradation induced by exocytosis of lysosome-related organelles and release of their cargo. Notably, an intermediate in nitric oxide synthesis, NG-hydroxy-l-arginine, shows efficacy in curtailing exocytosis of these organelles and improvement in animal survival. These data not only depict a novel mechanism responsible for very early glycocalyx degradation, but may also outline a potential preventive therapy. The second issue discussed in this article is related to the therapeutic acceleration of restoration of already degraded endothelial glycocalyx. Here, using as an example our recent findings obtained with sulodexide, we illustrate the importance of the expedited repair of degraded endothelial glycocalyx for the survival of animals with severe sepsis. These two focal points of the review on glycocalyx may not only have broader disease applicability, but they may also provide additional evidence to buttress the idea of the importance of endothelial glycocalyx and its maintenance and repair in the prevention and treatment of an array of renal and nonrenal diseases.

Endothelial cell dysfunction and glycocalyx – A vicious circle

Dysfunctional endothelial cells are an essential contributor to the progression of diverse chronic cardiovascular, renal, and metabolic diseases. It manifests in impairment of nitric oxide-dependent vasorelaxation, vascular permeability, and leukocytes deterrent. While endothelial glycocalyx is known to regulate these functions, glycocalyx has been shown to be impaired in pathologic settings leading to endothelial dysfunction. Are these findings coincidental or are they indicative of a potential cooperation of the glycocalyx and the endothelium in inducing a dysfunctional phenotype? The main thrust of this overview is to advance a hypothesis on the existence of vicious circle relations between impaired endothelial glycocalyx and endothelial cell dysfunction. We briefly introduce physiology and pathology of blood flow-induced components of mechanotransduction in endothelial cells, as this function is dependent on glycocalyx and is critically involved in the development of endothelial dysfunction. Next, we present a series of experimental findings and arguments favoring the view on the impairment of mechanotransduction in dysfunctional endothelia. We advance the concept of feedback reinforcement between perturbed endothelial glycocalyx and progression of endothelial dysfunction and sketch therapeutic approaches to restore them. Among those we introduce our recently designed liposomal nanocarriers of preassembled glycocalyx and present evidence of their ability to expeditiously restore endothelial mechanotransduction.