PLAC8-Mediated Activation of NOX4 Signalling Restores Angiogenic Function of Endothelial Colony-Forming Cells in Experimental Hypoxia.

Ischaemic cardiovascular disease is associated with tissue hypoxia as a significant determinant of angiogenic dysfunction and adverse remodelling. While cord blood-derived endothelial colony-forming cells (CB-ECFCs) hold clear therapeutic potential due to their enhanced angiogenic and proliferative capacity, their impaired functionality within the disease microenvironment represents a major barrier to clinical translation. The aim of this study was to define the specific contribution of NOX4 NADPH oxidase, which we previously reported as a key CB-ECFC regulator, to hypoxia-induced dysfunction and its potential as a therapeutic target. CB-ECFCs exposed to experimental hypoxia demonstrated downregulation of NOX4-mediated reactive oxygen species (ROS) signalling linked with a reduced tube formation, which was partially restored by NOX4 plasmid overexpression. siRNA knockdown of placenta-specific 8 (PLAC8), identified by microarray analysis as an upstream regulator of NOX4 in hypoxic versus normoxic CB-ECFCs, enhanced tube formation, NOX4 expression and hydrogen peroxide generation, and induced several key transcription factors associated with downstream Nrf2 signalling. Taken together, these findings indicated that activation of the PLAC8-NOX4 signalling axis improved CB-ECFC angiogenic functions in experimental hypoxia, highlighting this pathway as a potential target for protecting therapeutic cells against the ischaemic cardiovascular disease microenvironment.

GREM1 signaling in cancer: tumor promotor and suppressor?

GREMLIN1 (GREM1) is member of a family of structurally and functionally related secreted cysteine knot proteins, which act to sequester and inhibit the action of multifunctional bone morphogenetic proteins (BMPs). GREM1 binds directly to BMP dimers, thereby preventing BMP-mediated activation of BMP type I and type II receptors. Multiple reports identify the overexpression of GREM1 as a contributing factor in a broad range of cancers. Additionally, the GREM1 gene is amplified in a rare autosomal dominant inherited form of colorectal cancer. The inhibitory effects of GREM1 on BMP signaling have been linked to these tumor-promoting effects, including facilitating cancer cell stemness and the activation of cancer-associated fibroblasts. Moreover, GREM1 has been described to bind and signal to vascular endothelial growth factor receptor (VEGFR) and stimulate angiogenesis, as well as epidermal and fibroblast growth factor receptor (EGFR and FGFR) to elicit tumor-promoting effects in breast and prostate cancer, respectively. In contrast, a 2022 report revealed that GREM1 can promote an epithelial state in pancreatic cancers, thereby inhibiting pancreatic tumor growth and metastasis. In this commentary, we will review these disparate findings and attempt to provide clarity around the role of GREM1 signaling in cancer.

Mesenchymal stromal cells-derived extracellular vesicles reprogramme macrophages in ARDS models through the miR-181a-5p-PTEN-pSTAT5-SOCS1 axis.

RATIONALE: A better understanding of the mechanism of action of mesenchymal stromal cells (MSCs) and their extracellular vesicles (EVs) is needed to support their use as novel therapies for acute respiratory distress syndrome (ARDS). Macrophages are important mediators of ARDS inflammatory response. Suppressor of cytokine signalling (SOCS) proteins are key regulators of the macrophage phenotype switch. We therefore investigated whether SOCS proteins are involved in mediation of the MSC effect on human macrophage reprogramming. METHODS: Human monocyte-derived macrophages (MDMs) were stimulated with lipopolysaccharide (LPS) or plasma samples from patients with ARDS (these samples were previously classified into hypo-inflammatory and hyper-inflammatory phenotype) and treated with MSC conditioned medium (CM) or EVs. Protein expression was measured by Western blot. EV micro RNA (miRNA) content was determined by miRNA sequencing. In vivo: LPS-injured C57BL/6 mice were given EVs isolated from MSCs in which miR-181a had been silenced by miRNA inhibitor or overexpressed using miRNA mimic. RESULTS: EVs were the key component of MSC CM responsible for anti-inflammatory modulation of human macrophages. EVs significantly reduced secretion of tumour necrosis factor-α and interleukin-8 by LPS-stimulated or ARDS plasma-stimulated MDMs and this was dependent on SOCS1. Transfer of miR-181a in EVs downregulated phosphatase and tensin homolog (PTEN) and subsequently activated phosphorylated signal transducer and activator of transcription 5 (pSTAT5) leading to upregulation of SOCS1 in macrophages. In vivo, EVs alleviated lung injury and upregulated pSTAT5 and SOCS1 expression in alveolar macrophages in a miR181-dependent manner. Overexpression of miR-181a in MSCs significantly enhanced therapeutic efficacy of EVs in this model. CONCLUSION: miR-181a-PTEN-pSTAT5-SOCS1 axis is a novel pathway responsible for immunomodulatory effect of MSC EVs in ARDS.

Single-cell multiomics reveals the complexity of TGFß signalling to chromatin in iPSC-derived kidney organoids.

TGFß1 plays a regulatory role in the determination of renal cell fate and the progression of renal fibrosis. Here we show an association between SMAD3 and the histone methyltransferase, EZH2, during cell differentiation; ChIP-seq revealed that SMAD3 and EZH2 co-occupy the genome in iPSCs and in iPSC-derived nephron progenitors. Through integration of single cell gene expression and epigenome profiling, we identified de novo ACTA2+ve/POSTN+ve myofibroblasts in kidney organoids treated with TGFß1, characterised by increased SMAD3-dependent cis chromatin accessibility and gene expression associated with fibroblast activation. We have identified fibrosis-associated regulons characterised by enrichment of SMAD3, AP1, the ETS family of transcription factors, and NUAK1, CREB3L1, and RARG, corresponding to enriched motifs at accessible loci identified by scATACseq. Treatment with the EZH2 specific inhibitor GSK343, blocked SMAD3-dependent cis co-accessibility and inhibited myofibroblast activation. This mechanism, through which TGFß signals directly to chromatin, represents a critical determinant of fibrotic, differentiated states.

Loss of TRPV2-mediated blood flow autoregulation recapitulates diabetic retinopathy in rats.

Loss of retinal blood flow autoregulation is an early feature of diabetes that precedes the development of clinically recognizable diabetic retinopathy (DR). Retinal blood flow autoregulation is mediated by the myogenic response of the retinal arterial vessels, a process that is initiated by the stretch­dependent activation of TRPV2 channels on the retinal vascular smooth muscle cells (VSMCs). Here, we show that the impaired myogenic reaction of retinal arterioles from diabetic animals is associated with a complete loss of stretch­dependent TRPV2 current activity on the retinal VSMCs. This effect could be attributed, in part, to TRPV2 channel downregulation, a phenomenon that was also evident in human retinal VSMCs from diabetic donors. We also demonstrate that TRPV2 heterozygous rats, a nondiabetic model of impaired myogenic reactivity and blood flow autoregulation in the retina, develop a range of microvascular, glial, and neuronal lesions resembling those observed in DR, including neovascular complexes. No overt kidney pathology was observed in these animals. Our data suggest that TRPV2 dysfunction underlies the loss of retinal blood flow autoregulation in diabetes and provide strong support for the hypothesis that autoregulatory deficits are involved in the pathogenesis of DR.

Comparisons of α2-Adrenergic Agents, Medetomidine and Xylazine, with Pentobarbital for Anesthesia: Important Pitfalls in Diabetic and Nondiabetic Rats.

Purpose: Anesthesia is necessary to conduct rodent electroretinograms (ERGs). We evaluated utility of the α2-agonist medetomidine versus xylazine for ERG studies in nondiabetic and diabetic rats. Pentobarbital was included as a comparator. Methods: Male Sprague-Dawley rats, with and without streptozotocin (STZ)-induced diabetes, were anesthetized with medetomidine (1 mg/kg), xylazine (10 mg/kg) (both with ketamine 75 mg/kg), or pentobarbital (70 mg/kg). The depth of anesthesia was assessed, and if adequate, scotopic ERGs were recorded. Blood glucose was monitored. Results: In nondiabetic rats, all three agents induced satisfactory anesthesia, but with differing durations: medetomidine > pentobarbital > xylazine. ERG responses were similar under medetomidine and xylazine, but relatively reduced under pentobarbital. Both α2-agonists (but not pentobarbital) elicited marked hyperglycemia (peak values 316.1 ± 42.6 and 300.3 ± 29.5 mg/dL, respectively), persisting for 12 h. In diabetic rats, elevated blood glucose concentrations were not affected by any of the agents, but the depth of anesthesia under medetomidine and xylazine was inadequate for ERG recording. Conclusions: In nondiabetic rats, medetomidine and xylazine elicited comparable effects on ERGs that differ from pentobarbital, but both perturbed glucose metabolism, potentially confounding experimental outcomes. In STZ-diabetic rats, neither α2-agent provided adequate anesthesia, while pentobarbital did so. Problems with α2-anesthetic agents, including medetomidine, must be recognized to ensure meaningful interpretation of experimental results.

Effects of modified lipoproteins on human trophoblast cells: a role in pre-eclampsia in pregnancies complicated by diabetes.

INTRODUCTION: Pre-eclampsia (PE) is increased ~4-fold by maternal diabetes. Elevated plasma antiangiogenic factors, soluble fms-like tyrosine kinase (sFLT-1) and soluble endoglin (sENG), precede PE onset. We investigated whether diabetes-related stresses, modified lipoproteins and elevated glucose enhance trophoblast sFLT-1 and sENG release and/or alter placental barrier function and whether oxidized low-density lipoprotein (Ox-LDL) is in placental tissue. RESEARCH DESIGN AND METHODS: HTR8/SVneo cells were exposed to 'heavily-oxidized, glycated' LDL (HOG-LDL) versus native LDL (N-LDL) (10-200 mg protein/L) for 24 hours ±pretreatment with glucose (30 mmol/L, 72 hours). Concentrations of sFLT-1 and sENG in supernatants (by ELISA) and expressions of sFLT-1-I13 and sFLT-1-E15A isoforms, endoglin (ENG) and matrix metalloproteinase-14 (MMP-14; by RT-PCR) were quantified. For barrier studies, JAR cells were cultured in Transwell plates (12-14 days), then exposed to LDL. Transepithelial electrical resistance (TEER) was measured after 6, 12 and 24 hours. In placental sections from women with and without type 1 diabetes, immunostaining of apolipoprotein B100 (ApoB, a marker of LDL), Ox-LDL and lipoxidation product 4-hydroxynonenal was performed. RESULTS: HOG-LDL (50 mg/L) increased sFLT-1 (2.7-fold, p<0.01) and sENG (6.4-fold, p<0.001) in supernatants versus N-LDL. HOG-LDL increased expression of sFLT-1-I13 (twofold, p<0.05), sFLT-1-E15A (1.9-fold, p<0.05), ENG (1.6-fold, p<0.01) and MMP-14 (1.8-fold, p<0.05) versus N-LDL. High glucose did not by itself alter sFLT-1 or sENG concentrations, but potentiated effects of HOG-LDL on sFLT-1 by 1.5-fold (p<0.05) and on sENG by 1.8-fold (p<0.01). HOG-LDL (200 mg/L) induced trophoblast barrier impairment, decreasing TEER at 6 hours (p<0.01), 12 hours (p<0.01) and 24 hours (p<0.05) versus N-LDL. Immunostaining of term placental samples from women both with and without diabetes revealed presence of intravillous modified lipoproteins. CONCLUSION: These findings may explain, in part, the high risk for PE in women with diabetes. The trophoblast culture model has potential for evaluating novel therapies targeting barrier dysfunction.

The Potential of Albuminuria as a Biomarker of Diabetic Complications.

Diabetes mellitus is a disease of dysregulated blood glucose homeostasis. The current pandemic of diabetes is a significant driver of patient morbidity and mortality, as well as a major challenge to healthcare systems worldwide. The global increase in the incidence of diabetes has prompted researchers to focus on the different pathogenic processes responsible for type 1 and type 2 diabetes. Similarly, increased morbidity due to diabetic complications has accelerated research to uncover pathological changes causing these secondary complications. Albuminuria, or protein in the urine, is a well-recognised biomarker and risk factor for renal and cardiovascular disease. Albuminuria is a mediator of pathological abnormalities in diabetes-associated conditions such as nephropathy and atherosclerosis. Clinical screening and diagnosis of diabetic nephropathy is chiefly based on the presence of albuminuria. Given the ease in measuring albuminuria, the potential of using albuminuria as a biomarker of cardiovascular diseases is gaining widespread interest. To assess the benefits of albuminuria as a biomarker, it is important to understand the association between albuminuria and cardiovascular disease. This review examines our current understanding of the pathophysiological mechanisms involved in both forms of diabetes, with specific focus on the link between albuminuria and specific vascular complications of diabetes.

Secreted BMP antagonists and their role in cancer and bone metastases.

Bone morphogenetic proteins (BMPs) are multifunctional secreted cytokines that act in a highly context-dependent manner. BMP action extends beyond the induction of cartilage and bone formation, to encompass pivotal roles in controlling tissue and organ homeostasis during development and adulthood. BMPs signal via plasma membrane type I and type II serine/threonine kinase receptors and intracellular SMAD transcriptional effectors. Exquisite temporospatial control of BMP/SMAD signalling and crosstalk with other cellular cues is achieved by a series of positive and negative regulators at each step in the BMP/SMAD pathway. The interaction of BMP ligand with its receptors is carefully controlled by a diverse set of secreted antagonists that bind BMPs and block their interaction with their cognate BMP receptors. Perturbations in this BMP/BMP antagonist balance are implicated in a range of developmental disorders and diseases, including cancer. Here, we provide an overview of the structure and function of secreted BMP antagonists, and summarize recent novel insights into their role in cancer progression and bone metastasis. Gremlin1 (GREM1) is a highly studied BMP antagonist, and we will focus on this molecule in particular and its role in cancer. The therapeutic potential of pharmacological inhibitors for secreted BMP antagonists for cancer and other human diseases will also be discussed.

No evidence of Gremlin1-mediated activation of VEGFR2 signaling in endothelial cells.

Canonical Gremlin1 (GREM1) signaling involves binding to and sequestering bone morphogenetic proteins (BMPs) in the extracellular matrix, preventing the activation of cognate BMP receptor. Exquisite temporospatial control of the GREM1-BMP interaction is required during development, and perturbation of this balance leads to abnormal limb formation and defective kidney development. In addition to inhibition of BMP signaling, several other noncanonical signaling modalities of GREM1 have been postulated. Some literature reports have suggested that GREM1 can bind to and activate vascular endothelial growth factor receptor-2 (VEGFR2) in endothelial cells, human kidney epithelial cells, and others. These reports suggest that the GREM1 → VEGFR2 signaling can drive angiogenesis both in vitro and in vivo We report here that, despite exhaustive attempts, we did not observe GREM1 activation of VEGFR2 in any of the cell lines reported by the above-mentioned studies. Incubation of endothelial colony-forming cells (ECFCs) or human umbilical vein endothelial cells (HUVECs) with recombinant VEGF triggered a robust increase in VEGFR2 tyrosine phosphorylation. In contrast, no VEGFR2 phosphorylation was detected when cells were incubated with recombinant GREM1 over a range of time points and concentrations. We also show that GREM1 does not interfere with VEGF-mediated VEGFR2 activation, suggesting that GREM1 does not bind with any great affinity to VEGFR2. Measurements of ECFC barrier integrity revealed that VEGF induces barrier function disruption, but recombinant human GREM1 had no effect in this assay. We believe that these results provide an important clarification of the potential interaction between GREM1 and VEGFR2 in mammalian cells.

Uncoupled turnover disrupts mitochondrial quality control in diabetic retinopathy.

Mitochondrial quality control (MQC) is crucial for regulating CNS homeostasis, and its disruption has been implicated in the pathogenesis of some of the most common neurodegenerative diseases. In healthy tissues, the maintenance of MQC depends upon an exquisite balance between mitophagy (removal of damaged mitochondria by autophagy) and biogenesis (de novo synthesis of mitochondria). Here, we show that mitophagy is disrupted in diabetic retinopathy (DR) and decoupled from mitochondrial biogenesis during the progression of the disease. Diabetic retinas from human postmortem donors and experimental mice exhibit a net loss of mitochondrial contents during the early stages of the disease process. Using diabetic mitophagy-reporter mice (mitoQC-Ins2Akita) alongside pMitoTimer (a molecular clock to address mitochondrial age dynamics), we demonstrate that mitochondrial loss arose due to an inability of mitochondrial biogenesis to compensate for diabetes-exacerbated mitophagy. However, as diabetes duration increases, Pink1-dependent mitophagy deteriorates, leading to the build-up of mitochondria primed for degradation in DR. Impairment of mitophagy during prolonged diabetes is linked with the development of retinal senescence, a phenotype that blunted hyperglycemia-induced mitophagy in mitoQC primary Müller cells. Our findings suggest that normalizing mitochondrial turnover may preserve MQC and provide therapeutic options for the management of DR-associated complications.

Fibroblast-derived Gremlin1 localises to epithelial cells at the base of the intestinal crypt.

Gremlin1 (GREM1) is a secreted glycoprotein member of the differential screening-selected gene in aberrant neuroblastoma (DAN) family of bone morphogenetic protein (BMP) antagonists, which binds to BMPs preventing their receptor engagement. Previous studies have identified that stage II colorectal cancer (CRC) patients with high levels of GREM1 gene expression in their tumour tissue have a poorer prognosis. Using a series of in silico and in situ methodologies, we demonstrate that GREM1 gene expression is significantly higher (p < 0.0001) in CRC consensus molecular subtype 4 (CMS4), compared to the other CMS subtypes and correlates (p < 0.0001) with levels of cancer-associated fibroblasts (CAFs) within the CRC tumour microenvironment (TME). Our optimised immunohistochemistry protocol identified endogenous GREM1 protein expression in both the muscularis mucosa and adjacent colonic crypt bases in mouse intestine, in contrast to RNA expression which was shown to localise specifically to the muscularis mucosa, as determined by in situ hybridisation. Importantly, we demonstrate that cells with high levels of GREM1 expression display low levels of phospho-Smad1/5, consistent with reduced BMP signalling. Taken together, these data highlight a novel paracrine signalling circuit, which involves uptake of mature GREM1 protein by colonic crypt cells following secretion from neighbouring fibroblasts in the TME.

The vasoreparative potential of endothelial colony-forming cells in the ischemic retina is enhanced by cibinetide, a non-hematopoietic erythropoietin mimetic.

PURPOSE: Retinal ischemia remains a common sight threatening end-point in blinding diseases such as diabetic retinopathy and retinopathy of prematurity. Endothelial colony forming cells (ECFCs) represent a subpopulation of endothelial progenitors with therapeutic utility for promoting reparative angiogenesis in the ischaemic retina. The current study has investigated the potential of enhancing this cell therapy approach by the dampening of the pro-inflammatory milieu typical of ischemic retina. Based on recent findings that ARA290 (cibinetide), a peptide based on the Helix-B domain of erythropoietin (EPO), is anti-inflammatory and tissue-protective, the effect of this peptide on ECFC-mediated vascular regeneration was studied in the ischemic retina. METHODS: The effects of ARA290 on pro-survival signaling and function were assessed in ECFC cultures in vitro. Efficacy of ECFC transplantation therapy to promote retinal vascular repair in the presence and absence of ARA290 was studied in the oxygen induced retinopathy (OIR) model of retinal ischemia. The inflammatory cytokine profile and microglial activation were studied as readouts of inflammation. RESULTS: ARA290 activated pro-survival signaling and enhanced cell viability in response to H2O2-mediated oxidative stress in ECFCs in vitro. Preconditioning of ECFCs with EPO or ARA290 prior to delivery to the ischemic retina did not enhance vasoreparative function. ARA290 delivered systemically to OIR mice reduced pro-inflammatory expression of IL-1ß and TNF-α in the mouse retina. Following intravitreal transplantation, ECFCs incorporated into the damaged retinal vasculature and significantly reduced avascular area. The vasoreparative function of ECFCs was enhanced in the presence of ARA290 but not EPO. DISCUSSION: Regulation of the pro-inflammatory milieu of the ischemic retina can be enhanced by ARA290 and may be a useful adjunct to ECFC-based cell therapy for ischemic retinopathies.

CAMKII as a therapeutic target for growth factor-induced retinal and choroidal neovascularization.

While anti-VEGF drugs are commonly used to inhibit pathological retinal and choroidal neovascularization, not all patients respond in an optimal manner. Mechanisms underpinning resistance to anti­VEGF therapy include the upregulation of other proangiogenic factors. Therefore, therapeutic strategies that simultaneously target multiple growth factor signaling pathways would have significant value. Here, we show that Ca2+/calmodulin-dependent kinase II (CAMKII) mediates the angiogenic actions of a range of growth factors in human retinal endothelial cells and that this kinase acts as a key nodal point for the activation of several signal transduction cascades that are known to play a critical role in growth factor-induced angiogenesis. We also demonstrate that endothelial CAMKIIγ and -δ isoforms differentially regulate the angiogenic effects of different growth factors and that genetic deletion of these isoforms suppresses pathological retinal and choroidal neovascularization in vivo. Our studies suggest that CAMKII could provide a novel and efficacious target to inhibit multiple angiogenic signaling pathways for the treatment of vasoproliferative diseases of the eye. CAMKIIγ represents a particularly promising target, as deletion of this isoform inhibited pathological neovascularization, while enhancing reparative angiogenesis in the ischemic retina.

The roles of TRIO and F-actin-binding protein in glioblastoma cells.

TRIO and F-actin-binding protein (TrioBP), which was initially discovered as a binding partner of Trio and F­actin, is a critical factor associated with hearing loss in humans. However, the function of TrioBP in cancer has not been investigated. In the present study, TrioBP expression was indicated to be highly elevated in U87­MG and U343­MG cells. Furthermore, the TrioBP mRNA expression level was markedly increased in U87­MG and U251­MG cells compared with that in cerebral cortex cells, as determined by deep sequencing. Comprehensive analysis of a public TCGA dataset confirmed that TrioBP expression is elevated in patients with glioblastoma. In summary, the present data indicate that TrioBP expression is increased in glioblastoma cell lines and in patients with glioma, suggesting that TrioBP has potential as a diagnostic marker or therapeutic agent for glioma.

Modulation of PI3K/PTEN Pathway Does Not Affect Catalytic Activity of PDK1 in Jurkat Cells.

Unopposed phosphoinositide 3-kinase (PI3K) activity and 3-phosphoinositide production in Jurkat cells, due to a mutation in the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor-suppressor protein, results in deregulation of PH domain-containing proteins including the serine/threonine kinase PKB. In Jurkat cells, PKB is constitutively active and phosphorylated at the activation-loop residue (Thr308). 3-Phosphoinositide-dependent protein kinase-1 (PDK1), an enzyme that also contains a PH domain, catalyses Thr308 phosphorylation of PKB in addition to other kinase families such as PKC isoforms. It is unknown, however, whether the loss of PTEN in Jurkat cells also results in unregulated PDK1 activity and whether such loss has an impact on activation-loop phosphorylation of other PDK1 substrates e.g. PKC. In this study, we addressed whether loss of PTEN in Jurkat cells affects PDK1 catalytic activity and intracellular localization. We demonstrated that reducing the level of 3-phosphoinositides in Jurkat cells with pharmacological inhibitors of PI3K or expression of PTEN does not affect PDK1 activity or its intracellular localization. We conclude, therefore, that although Jurkat cells lack PTEN expression, only a subset of pathways downstream of PDK1 are perturbed as a consequence of PTEN loss.

Diabetic Nephropathy: a Tangled Web to Unweave.

Diabetic nephropathy (DN) is currently the leading cause of end-stage renal disease globally. Given the increasing incidence of diabetes, many experts hold the view that DN will eventually progress toward pandemic proportions. Whilst hyperglycaemia-induced vascular dysfunction is the primary initiating mechanism in DN, its progression is also driven by a heterogeneous set of pathological mechanisms, including oxidative stress, inflammation and fibrosis. Current treatment strategies for DN are targeted against the fundamental dysregulation of glycaemia and hypertension. Unfortunately, these standards of care can delay but do not prevent disease progression or the significant emotional, physical and financial costs associated with this disease. As such, there is a pressing need to develop novel therapeutics that are both effective and safe. Set against the genomic era, numerous potential target pathways in DN have been identified. However, the clinical translation of basic DN research has been met with a number of challenges. Moreover, the notion of DN as a purely vascular disease is outdated and it has become clear that DN is a multi-dimensional, multi-cellular condition. The review will highlight the current therapeutic approaches for DN and provide an insight into how the inherent complexity of DN is shaping the research pathways toward the development and clinical translation of novel therapeutic strategies.

Gremlin1 plays a key role in kidney development and renal fibrosis.

Gremlin1 (Grem1), an antagonist of bone morphogenetic proteins, plays a key role in embryogenesis. A highly specific temporospatial gradient of Grem1 and bone morphogenetic protein signaling is critical to normal lung, kidney, and limb development. Grem1 levels are increased in renal fibrotic conditions, including acute kidney injury, diabetic nephropathy, chronic allograft nephropathy, and immune glomerulonephritis. We demonstrate that a small number of grem1-/- whole body knockout mice on a mixed genetic background (8%) are viable, with a single, enlarged left kidney and grossly normal histology. The grem1-/- mice displayed mild renal dysfunction at 4 wk, which recovered by 16 wk. Tubular epithelial cell-specific targeted deletion of Grem1 (TEC-grem1-cKO) mice displayed a milder response in the acute injury and recovery phases of the folic acid model. Increases in indexes of kidney damage were smaller in TEC-grem1-cKO than wild-type mice. In the recovery phase of the folic acid model, associated with renal fibrosis, TEC-grem1-cKO mice displayed reduced histological damage and an attenuated fibrotic gene response compared with wild-type controls. Together, these data demonstrate that Grem1 expression in the tubular epithelial compartment plays a significant role in the fibrotic response to renal injury in vivo.

Hypoxia-induced responses by endothelial colony-forming cells are modulated by placental growth factor.

BACKGROUND: Endothelial colony-forming cells (ECFCs), also termed late outgrowth endothelial cells, are a well-defined circulating endothelial progenitor cell type with an established role in vascular repair. ECFCs have clear potential for cell therapy to treat ischaemic disease, although the precise mechanism(s) underlying their response to hypoxia remains ill-defined. METHODS: In this study, we isolated ECFCs from umbilical cord blood and cultured them on collagen. We defined the response of ECFCs to 1% O2 exposure at acute and chronic time points. RESULTS: In response to low oxygen, changes in ECFC cell shape, proliferation, size and cytoskeleton phenotype were detected. An increase in the number of senescent ECFCs also occurred as a result of long-term culture in 1% O2. Low oxygen exposure altered ECFC migration and tube formation in Matrigel®. Increases in angiogenic factors secreted from ECFCs exposed to hypoxia were also detected, in particular, after treatment with placental growth factor (PlGF). Exposure of cells to agents that stabilise hypoxia-inducible factors such as dimethyloxalylglycine (DMOG) also increased PlGF levels. Conditioned medium from both hypoxia-treated and DMOG-treated cells inhibited ECFC tube formation. This effect was reversed by the addition of PlGF neutralising antibody to the conditioned medium, confirming the direct role of PlGF in this effect. CONCLUSIONS: This study deepens our understanding of the response of ECFCs to hypoxia and also identifies a novel and important role for PlGF in regulating the vasculogenic potential of ECFCs.

IRS proteins and diabetic complications.

IRS proteins are cellular adaptor molecules that mediate many of the key metabolic actions of insulin. When tyrosine is phosphorylated by the activated insulin receptor, IRS proteins recruit downstream effectors, such as phosphoinositide 3-kinase and mitogen-activated protein kinase, in order to elicit cellular responses such as glucose uptake, lipid metabolism and cell proliferation. There are two main IRS proteins in humans (IRS1 and IRS2), both of which are widely expressed. Given their central role in the insulin signalling pathway, it is not surprising that male mice lacking Irs1 or Irs2 present with elevated blood glucose or type 2 diabetes, respectively. For reasons yet to be identified, female Irs2 (-/-) mice do not develop type 2 diabetes. A number of organs are affected by complications of diabetes; macrovascular complications include stroke and coronary artery disease, while nephropathy, neuropathy and retinopathy fall into the category of microvascular complications. Given the serious consequences of these complications on patient morbidity and mortality, it is essential to identify the molecular pathogenesis underlying diabetic complications, with a view to improving therapeutic intervention and patient outcomes. A number of recently published papers have converged on the hypothesis that the loss of insulin signalling and IRS proteins is instrumental to the development and/or progression of diabetic complications. This review will summarise some highlights from the published work in which this hypothesis is discussed.