Kidney repair and regeneration: perspectives of the NIDDK (Re)Building a Kidney consortium.

Acute kidney injury impacts âˆ¼13.3 million individuals and causes âˆ¼1.7 million deaths per year globally. Numerous injury pathways contribute to acute kidney injury, including cell cycle arrest, senescence, inflammation, mitochondrial dysfunction, and endothelial injury and dysfunction, and can lead to chronic inflammation and fibrosis. However, factors enabling productive repair versus nonproductive, persistent injury states remain less understood. The (Re)Building a Kidney (RBK) consortium is a National Institute of Diabetes and Digestive and Kidney Diseases consortium focused on both endogenous kidney repair mechanisms and the generation of new kidney tissue. This short review provides an update on RBK studies of endogenous nephron repair, addressing the following questions: (i) What is productive nephron repair? (ii) What are the cellular sources and drivers of repair? and (iii) How do RBK studies promote development of therapeutics? Also, we provide a guide to RBK's open access data hub for accessing, downloading, and further analyzing data sets.

The heparin binding domain of von Willebrand factor binds to growth factors and promotes angiogenesis in wound healing.

During wound healing, the distribution, availability, and signaling of growth factors (GFs) are orchestrated by their binding to extracellular matrix components in the wound microenvironment. Extracellular matrix proteins have been shown to modulate angiogenesis and promote wound healing through GF binding. The hemostatic protein von Willebrand factor (VWF) released by endothelial cells (ECs) in plasma and in the subendothelial matrix has been shown to regulate angiogenesis; this function is relevant to patients in whom VWF deficiency or dysfunction is associated with vascular malformations. Here, we show that VWF deficiency in mice causes delayed wound healing accompanied by decreased angiogenesis and decreased amounts of angiogenic GFs in the wound. We show that in vitro VWF binds to several GFs, including vascular endothelial growth factor-A (VEGF-A) isoforms and platelet-derived growth factor-BB (PDGF-BB), mainly through the heparin-binding domain (HBD) within the VWF A1 domain. VWF also binds to VEGF-A and fibroblast growth factor-2 (FGF-2) in human plasma and colocalizes with VEGF-A in ECs. Incorporation of the VWF A1 HBD into fibrin matrices enables sequestration and slow release of incorporated GFs. In vivo, VWF A1 HBD-functionalized fibrin matrices increased angiogenesis and GF retention in VWF-deficient mice. Treatment of chronic skin wounds in diabetic mice with VEGF-A165 and PDGF-BB incorporated within VWF A1 HBD-functionalized fibrin matrices accelerated wound healing, with increased angiogenesis and smooth muscle cell proliferation. Therefore, the VWF A1 HBD can function as a GF reservoir, leading to effective angiogenesis and tissue regeneration.

A brief exposure to tryptase or thrombin potentiates fibrocyte differentiation in the presence of serum or serum amyloid p.

A key question in both wound healing and fibrosis is the trigger for the initial formation of scar tissue. To help form scar tissue, circulating monocytes enter the tissue and differentiate into fibroblast-like cells called fibrocytes, but fibrocyte differentiation is strongly inhibited by the plasma protein serum amyloid P (SAP), and healthy tissues contain very few fibrocytes. In wounds and fibrotic lesions, mast cells degranulate to release tryptase, and thrombin mediates blood clotting in early wounds. Tryptase and thrombin are upregulated in wound healing and fibrotic lesions, and inhibition of these proteases attenuates fibrosis. We report that tryptase and thrombin potentiate human fibrocyte differentiation at biologically relevant concentrations and exposure times, even in the presence of concentrations of serum and SAP that normally completely inhibit fibrocyte differentiation. Fibrocyte potentiation by thrombin and tryptase is mediated by protease-activated receptors 1 and 2, respectively. Together, these results suggest that tryptase and thrombin may be an initial trigger to override SAP inhibition of fibrocyte differentiation to initiate scar tissue formation.

Galectin-3 Binding Protein Secreted by Breast Cancer Cells Inhibits Monocyte-Derived Fibrocyte Differentiation.

To metastasize, tumor cells often need to migrate through a layer of collagen-containing scar tissue which encapsulates the tumor. A key component of scar tissue and fibrosing diseases is the monocyte-derived fibrocyte, a collagen-secreting profibrotic cell. To test the hypothesis that invasive tumor cells may block the formation of the fibrous sheath, we determined whether tumor cells secrete factors that inhibit monocyte-derived fibrocyte differentiation. We found that the human metastatic breast cancer cell line MDA-MB-231 secretes activity that inhibits human monocyte-derived fibrocyte differentiation, whereas less aggressive breast cancer cell lines secrete less of this activity. Purification indicated that Galectin-3 binding protein (LGALS3BP) is the active factor. Recombinant LGALS3BP inhibits monocyte-derived fibrocyte differentiation, and immunodepletion of LGALS3BP from MDA-MB 231 conditioned media removes the monocyte-derived fibrocyte differentiation-inhibiting activity. LGALS3BP inhibits the differentiation of monocyte-derived fibrocytes from wild-type mouse spleen cells, but not from SIGN-R1(-/-) mouse spleen cells, suggesting that CD209/SIGN-R1 is required for the LGALS3BP effect. Galectin-3 and galectin-1, binding partners of LGALS3BP, potentiate monocyte-derived fibrocyte differentiation. In breast cancer biopsies, increased levels of tumor cell-associated LGALS3BP were observed in regions of the tumor that were invading the surrounding stroma. These findings suggest LGALS3BP and galectin-3 as new targets to treat metastatic cancer and fibrosing diseases.

A retinoblastoma orthologue is required for the sensing of a chalone in Dictyostelium discoideum.

Retinoblastoma-like proteins regulate cell differentiation and inhibit cell proliferation. The Dictyostelium discoideum retinoblastoma orthologue RblA affects the differentiation of cells during multicellular development, but it is unclear whether RblA has a significant effect on Dictyostelium cell proliferation, which is inhibited by the secreted proteins AprA and CfaD. We found that rblA⁻ cells in shaking culture proliferate to a higher density, die faster after reaching stationary density, and, after starvation, have a lower spore viability than wild-type cells, possibly because in shaking culture, rblA⁻ cells have both increased cytokinesis and lower extracellular accumulation of CfaD. However, rblA⁻ cells have abnormally slow proliferation on bacterial lawns. Recombinant AprA inhibits the proliferation of wild-type cells but not that of rblA⁻ cells, whereas CfaD inhibits the proliferation of both wild-type cells and rblA⁻ cells. Similar to aprA⁻ cells, rblA⁻ cells have a normal mass and protein accumulation rate on a per-nucleus basis, indicating that RblA affects cell proliferation but not cell growth. AprA also functions as a chemorepellent, and RblA is required for proper AprA chemorepellent activity despite the fact that RblA does not affect cell speed. Together, our data indicate that an autocrine proliferation-inhibiting factor acts through RblA to regulate cell density in Dictyostelium, suggesting that such factors may signal through retinoblastoma-like proteins to control the sizes of structures such as developing organs or tumors.

Therapeutic use of α2-antiplasmin as an antifibrinolytic and hemostatic agent in surgery and regenerative medicine.

The biomaterial fibrin is widely used as a clinical tissue sealant in surgery. In preclinical research, fibrin is also extensively studied as a carrier material for growth factor delivery. In these applications, premature fibrin degradation leads to recurrent bleeding, tissue dehiscence and limited regenerative efficacy. Therefore, fibrinolysis inhibitors have been added to clinical fibrin formulations, for example the bovine-derived serine protease inhibitor aprotinin. Aprotinin is additionally used as a hemostatic agent to prevent excessive bleeding during surgery, in this case protecting endogenous fibrin clots. Nevertheless, aprotinin use has been associated with serious safety issues. Here, we explore the use the human physiological fibrinolysis inhibitor α2-antiplasmin (α2PI) as a substitute for aprotinin. We evaluate the efficacy of α2PI in the three main applications of aprotinin. We first showed that recombinant α2PI can successfully prolong the durability of fibrin biomaterials as compared to aprotinin in a model of subcutaneous implantation in mice mimicking application as a tissue sealant. We then used α2PI to enhance the delivery of engineered vascular endothelial growth factor (VEGF)-A and platelet-derived growth factor (PDGF)-BB in fibrin in promoting diabetic wound healing, which lead to improved wound closure, granulation tissue formation and angiogenesis. Lastly, we demonstrated that α2PI can be as effective as aprotinin as an intravenous hemostatic agent to prevent blood loss, using a tail-vein bleeding model in mice. Therefore, we believe that engineering fibrin biomaterials or endogenous fibrin with α2PI can have a strong impact in surgery and regenerative medicine by providing a competitive substitute to aprotinin that is of human origin.

VEGF-A, PDGF-BB and HB-EGF engineered for promiscuous super affinity to the extracellular matrix improve wound healing in a model of type 1 diabetes.

Chronic non-healing wounds, frequently caused by diabetes, lead to lower quality of life, infection, and amputation. These wounds have limited treatment options. We have previously engineered growth factors to bind to exposed extracellular matrix (ECM) in the wound environment using the heparin-binding domain of placental growth factor-2 (PlGF-2123-144), which binds promiscuously to ECM proteins. Here, in the type 1 diabetic (T1D) NOD mouse model, engineered growth factors (eGFs) improved both re-epithelialization and granulation tissue formation. eGFs were even more potent in combination, and the "triple therapy" of vascular endothelial growth factor-A (VEGF-PlGF-2123-144), platelet-derived growth factor-BB (PDGF-BB-PlGF-2123-144), and heparin-binding epidermal growth factor (HB-EGF-PlGF-2123-144) both improved wound healing and remained at the site of administration for significantly longer than wild-type growth factors. In addition, we also found that changes in the cellular milieu of a wound, including changing amounts of M1 macrophages, M2 macrophages and effector T cells, are most predictive of wound-healing success in the NOD mouse model. These results suggest that the triple therapy of VEGF-PlGF-2123-144, PDGF-BB-PlGF-2123-144, and HB-EGF-PlGF-2123-144 may be an effective therapy for chronic non-healing wounds in that occur as a complication of diabetes.

Protease activated-receptor 2 is necessary for neutrophil chemorepulsion induced by trypsin, tryptase, or dipeptidyl peptidase IV.

Compared to neutrophil chemoattractants, relatively little is known about the mechanism neutrophils use to respond to chemorepellents. We previously found that the soluble extracellular protein dipeptidyl peptidase IV (DPPIV) is a neutrophil chemorepellent. In this report, we show that an inhibitor of the protease activated receptor 2 (PAR2) blocks DPPIV-induced human neutrophil chemorepulsion, and that PAR2 agonists such as trypsin, tryptase, 2f-LIGRL, SLIGKV, and AC55541 induce human neutrophil chemorepulsion. Several PAR2 agonists in turn block the ability of the chemoattractant fMLP to attract neutrophils. Compared to neutrophils from male and female C57BL/6 mice, neutrophils from male and female mice lacking PAR2 are insensitive to the chemorepulsive effects of DPPIV or PAR2 agonists. Acute respiratory distress syndrome (ARDS) involves an insult-mediated influx of neutrophils into the lungs. In a mouse model of ARDS, aspiration of PAR2 agonists starting 24 h after an insult reduce neutrophil numbers in the bronchoalveolar lavage (BAL) fluid, as well as the post-BAL lung tissue. Together, these results indicate that the PAR2 receptor mediates DPPIV-induced chemorepulsion, and that PAR2 agonists might be useful to induce neutrophil chemorepulsion.

Laminin heparin-binding peptides bind to several growth factors and enhance diabetic wound healing.

Laminin, as a key component of the basement membrane extracellular matrix (ECM), regulates tissue morphogenesis. Here, we show that multiple laminin isoforms promiscuously bind to growth factors (GFs) with high affinity, through their heparin-binding domains (HBDs) located in the α chain laminin-type G (LG) domains. These domains also bind to syndecan cell-surface receptors, promoting attachment of fibroblasts and endothelial cells. We explore the application of these multifunctional laminin HBDs in wound healing in the type-2 diabetic mouse. We demonstrate that covalent incorporation of laminin HBDs into fibrin matrices improves retention of GFs and significantly enhances the efficacy of vascular endothelial cell growth factor (VEGF-A165) and platelet-derived growth factor (PDGF-BB) in promoting wound healing in vivo, under conditions where the GFs alone in fibrin are inefficacious. This laminin HBD peptide may be clinically useful by improving biomaterial matrices as both GF reservoirs and cell scaffolds, leading to effective tissue regeneration.

Trypsin, Tryptase, and Thrombin Polarize Macrophages towards a Pro-Fibrotic M2a Phenotype.

For both wound healing and the formation of a fibrotic lesion, circulating monocytes enter the tissue and differentiate into fibroblast-like cells called fibrocytes and pro-fibrotic M2a macrophages, which together with fibroblasts form scar tissue. Monocytes can also differentiate into classically activated M1 macrophages and alternatively activated M2 macrophages. The proteases thrombin, which is activated during blood clotting, and tryptase, which is released by activated mast cells, potentiate fibroblast proliferation and fibrocyte differentiation, but their effect on macrophages is unknown. Here we report that thrombin, tryptase, and the protease trypsin bias human macrophage differentiation towards a pro-fibrotic M2a phenotype expressing high levels of galectin-3 from unpolarized monocytes, or from M1 and M2 macrophages, and that these effects appear to operate through protease-activated receptors. These results suggest that proteases can initiate scar tissue formation by affecting fibroblasts, fibrocytes, and macrophages.

Trypsin potentiates human fibrocyte differentiation.

Trypsin-containing topical treatments can be used to speed wound healing, although the mechanism of action is unknown. To help form granulation tissue and heal wounds, monocytes leave the circulation, enter the wound tissue, and differentiate into fibroblast-like cells called fibrocytes. We find that 20 to 200 ng/ml trypsin (concentrations similar to those used in wound dressings) potentiates the differentiation of human monocytes to fibrocytes in cell culture. Adding trypsin inhibitors increases the amount of trypsin needed to potentiate fibrocyte differentiation, suggesting that the potentiating effect is dependent on trypsin proteolytic activity. Proteases with other site specificities such as pepsin, endoprotease GluC, and chymotrypsin do not potentiate fibrocyte differentiation. This potentiation requires the presence of albumin in the culture medium, and tryptic fragments of human or bovine albumin also potentiate fibrocyte differentiation. These results suggest that topical trypsin speeds wound healing by generating tryptic fragments of albumin, which in turn potentiate fibrocyte differentiation.