Genetic reprogramming with stem cells regenerates glomerular epithelial podocytes in Alport syndrome.

Glomerular filtration relies on the type IV collagen (ColIV) network of the glomerular basement membrane, namely, in the triple helical molecules containing the α3, α4, and α5 chains of ColIV. Loss of function mutations in the genes encoding these chains (Col4a3, Col4a4, and Col4a5) is associated with the loss of renal function observed in Alport syndrome (AS). Precise understanding of the cellular basis for the patho-mechanism remains unknown and a specific therapy for this disease does not currently exist. Here, we generated a novel allele for the conditional deletion of Col4a3 in different glomerular cell types in mice. We found that podocytes specifically generate α3 chains in the developing glomerular basement membrane, and that its absence is sufficient to impair glomerular filtration as seen in AS. Next, we show that horizontal gene transfer, enhanced by TGFß1 and using allogenic bone marrow-derived mesenchymal stem cells and induced pluripotent stem cells, rescues Col4a3 expression and revive kidney function in Col4a3-deficient AS mice. Our proof-of-concept study supports that horizontal gene transfer such as cell fusion enables cell-based therapy in Alport syndrome.

The transcription factor Tcf21 is required for specifying Foxd1 cells to the juxtaglomerular cell lineage.

Renin is a crucial enzyme involved in the regulation of blood pressure and electrolyte balance. It has been shown that renin expressing cells arise from the Foxd1+ stromal progenitors, however the factors involved in guiding Foxd1+ cells towards the renin-secreting cell fate remain poorly understood. Tcf21, also known as Pod1 or Capsulin, is a bHLH transcription factor that is expressed in the metanephric mesenchyme and plays a crucial role in kidney development. We have previously shown that deletion of Tcf21 in Foxd1+ cells ( Foxd1 Cre/+ ;Tcf21 f/f ) results in paucity of vascular mural cells and in disorganized renal arterial tree with fewer, shorter, and thinner arterioles. Here, we sought to examine the relationship between Tcf21 and renin cells during kidney development and test whether Tcf21 is implicated in the regulation of juxtaglomerular cell differentiation. Immunostaining for renin demonstrated that kidneys of Foxd1 Cre/+ ;Tcf21 f/f have fewer renin-positive spots at E16.5 and E18.5 compared with controls. In-situ hybridization for renin mRNA showed reduced expression in Foxd1 Cre/+ ;Tcf21 f/f kidneys at E14.5, E16.5, and E18.5. Together, these data suggest that stromal expression of Tcf21 is required for the emergence of renin cells. To dissect the role of Tcf21 in juxtaglomerular (JG) cells, we deleted Tcf21 upon renin promoter activation ( Ren1 dCre/+ ;Tcf21 f/f ). Interestingly, the Ren1 dCre/+ ;Tcf21 f/f kidney showed normal arterial tree at E16.5 identical to controls. Furthermore, inactivation of Tcf21 upon renin expression did not alter kidney morphology in two- and four-month-old mice. Finally, expression renin mRNA was similar between Ren1 dCre/+ ;Tcf21 f/f and controls at 2 months. Taken together, these findings suggest that Tcf21 expression in Foxd1+ cells is essential for specifying the fate of these cells into juxtaglomerular cells. However, once renin cell identity is assumed, Tcf21 is dispensable. Uncovering the regulation of Foxd1+ cells and their derivatives, including the JG cell lineage, is crucial for understanding the mechanisms underlying renal vasculature formation.

Post-ischemic inactivation of HIF prolyl hydroxylases in endothelium promotes maladaptive kidney repair by inducing glycolysis.

Ischemic acute kidney injury (AKI) is common in hospitalized patients and increases the risk for chronic kidney disease (CKD). Impaired endothelial cell (EC) functions are thought to contribute in AKI to CKD transition, but the underlying mechanisms remain unclear. Here, we identify a critical role for endothelial oxygen sensing prolyl hydroxylase domain (PHD) enzymes 1-3 in regulating post-ischemic kidney repair. In renal endothelium, we observed compartment-specific differences in the expression of the three PHD isoforms in both mice and humans. We found that post-ischemic concurrent inactivation of endothelial PHD1, PHD2, and PHD3 but not PHD2 alone promoted maladaptive kidney repair characterized by exacerbated tissue injury, fibrosis, and inflammation. Single-cell RNA-seq analysis of the post-ischemic endothelial PHD1, PHD2 and PHD3 deficient (PHDTiEC) kidney revealed an endothelial glycolytic transcriptional signature, also observed in human kidneys with severe AKI. This metabolic program was coupled to upregulation of the SLC16A3 gene encoding the lactate exporter monocarboxylate transporter 4 (MCT4). Strikingly, treatment with the MCT4 inhibitor syrosingopine restored adaptive kidney repair in PHDTiEC mice. Mechanistically, MCT4 inhibition suppressed pro-inflammatory EC activation reducing monocyte-endothelial cell interaction. Our findings suggest avenues for halting AKI to CKD transition based on selectively targeting the endothelial hypoxia-driven glycolysis/MCT4 axis.

The Association of Intravitreal Anti-VEGF Injections With Kidney Function in Diabetic Retinopathy.

Purpose: To examine whether patients with diabetic retinopathy receiving intravitreal anti-VEGF injections are at increased risk of kidney function decline. Design: Retrospective cohort study. Participants: Included 187 patients who received intravitreal anti-VEGF injections for proliferative diabetic retinopathy (PDR) and/or diabetic macular edema (DME), and 929 controls with non-PDR who did not receive injections, at a large tertiary care center in Chicago, Illinois. Methods: We queried our institutional enterprise data warehouse to identify patients with diabetic retinopathy, determined whether they received intravitreal anti-VEGF injections, and followed kidney function for all patients over time. Main Outcome Measures: We assessed time to sustained 40% decline in estimated glomerular filtration rate (eGFR) from baseline in patients receiving intravitreal anti-VEGF injections and compared it with controls using Kaplan-Meier and multivariable adjusted Cox proportional hazards regression models. Results: This study included 1116 patients (565 female [50.6%]; mean [standard deviation {SD}] age, 57.3 [13.6] years; mean [SD] eGFR, 65.3 [32.1] ml/min/1.73 m2). Of these, 187 patients received ≥ 1 intravitreal anti-VEGF injection (mean [SD], 11.4 [13.1] injections) for PDR and/or DME, and 929 controls with non-PDR received no injections. Intravitreal anti-VEGF injection use was not associated with an increased risk of kidney function decline (hazard ratio [HR], 1.44; 95% confidence interval [CI], 0.97-2.15). Subgroup analyses revealed that use of intravitreal anti-VEGF injections was associated with increased risk of kidney function decline in male patients (HR, 1.87; 95% CI, 1.11-3.14) but not female patients (HR, 0.97; 95% CI, 0.50-1.89). Intravitreal anti-VEGF injection use was also associated with an increased risk of kidney function decline in patients with baseline eGFR > 30 ml/min/1.73 m2 (HR, 1.86; 95% CI, 1.15-3.01), but not in individuals with baseline eGFR ≤ 30 ml/min/1.73 m2 (HR, 0.97; 95% CI, 0.45-2.10). Among patients who received injections, receiving ≥ 12 injections was not associated with risk of kidney function decline (HR, 1.13; 95% CI, 0.52-2.49). Conclusions: Intravitreal anti-VEGF injections for patients with diabetic retinopathy are overall well-tolerated with respect to kidney function, but the use of intravitreal anti-VEGF injections was associated with an increased risk of kidney function decline in certain subgroups of patients. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Differential roles of FOXC2 in the trabecular meshwork and Schlemm's canal in glaucomatous pathology.

Impaired development and maintenance of Schlemm's canal (SC) are associated with perturbed aqueous humor outflow and intraocular pressure. The angiopoietin (ANGPT)/TIE2 signaling pathway regulates SC development and maintenance, whereas the molecular mechanisms of crosstalk between SC and the neural crest (NC)-derived neighboring tissue, the trabecular meshwork (TM), are poorly understood. Here, we show NC-specific forkhead box (Fox)c2 deletion in mice results in impaired SC morphogenesis, loss of SC identity, and elevated intraocular pressure. Visible-light optical coherence tomography analysis further demonstrated functional impairment of the SC in response to changes in intraocular pressure in NC-Foxc2 -/- mice, suggesting altered TM biomechanics. Single-cell RNA-sequencing analysis identified that this phenotype is predominately characterized by transcriptional changes associated with extracellular matrix organization and stiffness in TM cell clusters, including increased matrix metalloproteinase expression, which can cleave the TIE2 ectodomain to produce soluble TIE2. Moreover, endothelial-specific Foxc2 deletion impaired SC morphogenesis because of reduced TIE2 expression, which was rescued by deleting the TIE2 phosphatase VE-PTP. Thus, Foxc2 is critical in maintaining SC identity and morphogenesis via TM-SC crosstalk.

VEGF-C overexpression in kidney progenitor cells is a model of renal lymphangiectasia.

Background: Lymphangiogenesis is believed to be a protective response in the setting of multiple forms of kidney injury and mitigates the progression of interstitial fibrosis. To augment this protective response, promoting kidney lymphangiogenesis is being investigated as a potential treatment to slow the progression of kidney disease.As injury related lymphangiogenesis is driven by signaling from the receptor VEGFR-3 in response to the cognate growth factor VEGF-C released by tubular epithelial cells, this signaling pathway is a candidate for future kidney therapeutics. However, the consequences to kidney development and function to targeting this signaling pathway remains poorly defined. Methods: We generated a new mouse model expressing Vegf-C under regulation of the nephron progenitor Six2Cre driver strain (Six2Vegf-C). Mice underwent a detailed phenotypic evaluation. Whole kidneys were processed for histology and micro computed tomography 3-dimensional imaging. Results: Six2Vegf-C mice had reduced body weight and kidney function compared to littermate controls. Six2Vegf-C kidneys demonstrated large peripelvic fluid filled lesions with distortion of the pelvicalcyceal system which progressed in severity with age. 3D imaging showed a 3-fold increase in total cortical vascular density. Histology confirmed a substantial increase in LYVE1+/PDPN+/VEGFR3+ lymphatic capillaries extending alongside EMCN+ peritubular capillaries. There was no change in EMCN+ peritubular capillary density. Conclusions: Kidney lymphangiogenesis was robustly induced in the Six2Vegf-C mice. There were no changes in peritubular blood capillary density despite these endothelial cells also expressing VEGFR-3. The model resulted in a severe cystic kidney phenotype that resembled a human condition termed renal lymphangiectasia. This study defines the vascular consequences of augmenting VEGF-C signaling during kidney development and provides new insight into a mimicker of human cystic kidney disease.

Activation of Angiopoietin-Tie2 Signaling Protects the Kidney from Ischemic Injury by Modulation of Endothelial-Specific Pathways.

SIGNIFICANCE STATEMENT: Ischemia-reperfusion AKI (IR-AKI) is common and causes significant morbidity. Effective treatments are lacking. However, preclinical studies suggest that inhibition of angiopoietin-Tie2 vascular signaling promotes injury, whereas activation of Tie2 is protective. We show that kidney ischemia leads to increased levels of the endothelial-specific phosphatase vascular endothelial protein tyrosine phosphatase (VE-PTP; PTPRB), which inactivates Tie2. Activation of Tie2 through VE-PTP deletion, or delivery of a novel angiopoietin mimetic (Hepta-ANG1), abrogated IR-AKI in mice. Single-cell RNAseq analysis showed Tie2 activation promotes increased Entpd1 expression, downregulation of FOXO1 target genes in the kidney vasculature, and emergence of a new subpopulation of glomerular endothelial cells. Our data provide a molecular basis and identify a candidate therapeutic to improve endothelial integrity and kidney function after IR-AKI. BACKGROUND: Ischemia-reperfusion AKI (IR-AKI) is estimated to affect 2%-7% of all hospitalized patients. The significant morbidity and mortality associated with AKI indicates urgent need for effective treatments. Previous studies have shown activation of the vascular angiopoietin-Tie2 tyrosine kinase signaling pathway abrogates ischemia-reperfusion injury (IRI). We extended previous studies to (1) determine the molecular mechanism(s) underlying kidney injury and protection related to decreased or increased activation of Tie2, respectively, and (2) to test the hypothesis that deletion of the Tie2 inhibitory phosphatase vascular endothelial protein tyrosine phosphatase (VE-PTP) or injection of a new angiopoietin mimetic protects the kidney from IRI by common molecular mechanism(s). METHODS: Bilateral IR-AKI was performed in VE-PTP wild-type or knockout mice and in C57BL/6J mice treated with Hepta-ANG1 or vehicle. Histologic, immunostaining, and single-cell RNA sequencing analyses were performed. RESULTS: The phosphatase VE-PTP, which negatively regulates the angiopoietin-Tie2 pathway, was upregulated in kidney endothelial cells after IRI, and genetic deletion of VE-PTP in mice protected the kidney from IR-AKI. Injection of Hepta-ANG1 potently activated Tie2 and protected the mouse kidney from IRI. Single-cell RNAseq analysis of kidneys from Hepta-ANG1-treated and vehicle-treated mice identified endothelial-specific gene signatures and emergence of a new glomerular endothelial subpopulation associated with improved kidney function. Overlap was found between endothelial-specific genes upregulated by Hepta-ANG1 treatment and those downregulated in HUVECs with constitutive FOXO1 activation, including Entpd1 / ENTPD1 that modulates purinergic receptor signaling. CONCLUSIONS: Our data support a key role of the endothelium in the development of IR-AKI, introduce Hepta-ANG1 as a putative new therapeutic biologic, and report a model to explain how IRI reduces Tie2 signaling and how Tie2 activation protects the kidney. PODCAST: This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/JASN/2023_05_23_JSN_Ang_EP23_052323.mp3.

Formation of the glomerular microvasculature is regulated by VEGFR-3.

Microvascular dysfunction is a key driver of kidney disease. Pathophysiological changes in the kidney vasculature are regulated by vascular endothelial growth factor receptors (VEGFRs), supporting them as potential therapeutic targets. The tyrosine kinase receptor VEGFR-3, encoded by FLT4 and activated by the ligands VEGF-C and VEGF-D, is best known for its role in lymphangiogenesis. Therapeutically targeting VEGFR-3 to modulate lymphangiogenesis has been proposed as a strategy to treat kidney disease. However, outside the lymphatics, VEGFR-3 is also expressed in blood vascular endothelial cells in several tissues including the kidney. Here, we show that Vegfr-3 is expressed in fenestrated microvascular beds within the developing and adult mouse kidney, which include the glomerular capillary loops. We found that expression levels of VEGFR-3 are dynamic during glomerular capillary loop development, with the highest expression observed during endothelial cell migration into the S-shaped glomerular body. We developed a conditional knockout mouse model for Vegfr-3 and found that loss of Vegfr-3 resulted in a striking glomerular phenotype characterized by aneurysmal dilation of capillary loops, absence of mesangial structure, abnormal interendothelial cell junctions, and poor attachment between glomerular endothelial cells and the basement membrane. In addition, we demonstrated that expression of the VEGFR-3 ligand VEGF-C by podocytes and mesangial cells is dispensable for glomerular development. Instead, VEGFR-3 in glomerular endothelial cells attenuates VEGFR-2 phosphorylation. Together, the results of our study support a VEGF-C-independent functional role for VEGFR-3 in the kidney microvasculature outside of lymphatic vessels, which has implications for clinical therapies that target this receptor.NEW & NOTEWORTHY Targeting VEGFR-3 in kidney lymphatics has been proposed as a method to treat kidney disease. However, expression of VEGFR-3 is not lymphatic-specific. We demonstrated developmental expression of VEGFR-3 in glomerular endothelial cells, with loss of Vegfr-3 leading to malformation of glomerular capillary loops. Furthermore, we showed that VEGFR-3 attenuates VEGFR-2 activity in glomerular endothelial cells independent of paracrine VEGF-C signaling. Together, these data provide valuable information for therapeutic development targeting these pathways.

Chemical inhibition of oxygen-sensing prolyl hydroxylases impairs angiogenic competence of human vascular endothelium through metabolic reprogramming.

Endothelial cell (EC) metabolism has emerged as a driver of angiogenesis. While hypoxia inactivates the oxygen sensors prolyl-4 hydroxylase domain-containing proteins 1-3 (PHD1-3) and stimulates angiogenesis, the effects of PHDs on EC functions remain poorly defined. Here, we investigated the impact of chemical PHD inhibition by dimethyloxalylglycine (DMOG) on angiogenic competence and metabolism of human vascular ECs. DMOG reduced EC proliferation, migration, and tube formation capacities, responses that were associated with an unfavorable metabolic reprogramming. While glycolytic genes were induced, multiple genes encoding sub-units of mitochondrial complex I were suppressed with concurrent decline in nicotinamide adenine dinucleotide (NAD+) levels. Importantly, the DMOG-induced defects in EC migration could be partially rescued by augmenting NAD+ levels through nicotinamide riboside or citrate supplementation. In summary, by integrating functional assays, transcriptomics, and metabolomics, we provide insights into the effects of PHD inhibition on angiogenic competence and metabolism of human vascular ECs.

Angiopoietin-1 Is Required for Vortex Vein and Choriocapillaris Development in Mice.

BACKGROUND: The choroidal vasculature, including the choriocapillaris and vortex veins, is essential for providing nutrients to the metabolically demanding photoreceptors and retinal pigment epithelium. Choroidal vascular dysfunction leads to vision loss and is associated with age-related macular degeneration and the poorly understood pachychoroid diseases including central serous chorioretinopathy and polypoidal choroidal vasculopathy that are characterized by formation of dilated pachyvessels throughout the choroid. METHODS: Using neural crest-specific Angpt1 knockout mice, we show that Angiopoietin 1, a ligand of the endothelial receptor TEK (also known as Tie2) is essential for choriocapillaris development and vortex vein patterning. RESULTS: Lacking choroidal ANGPT1, neural crest-specific Angpt1 knockout eyes exhibited marked choriocapillaris attenuation and 50% reduction in number of vortex veins, with only 2 vortex veins present in the majority of eyes. Shortly after birth, dilated choroidal vessels resembling human pachyvessels were observed extending from the remaining vortex veins and displacing the choriocapillaris, leading to retinal pigment epithelium dysfunction and subretinal neovascularization similar to that seen in pachychoroid disease. CONCLUSIONS: Together, these findings identify a new role for ANGPT1 in ocular vascular development and demonstrate a clear link between vortex vein dysfunction, pachyvessel formation, and disease.

Stromal Transcription Factor 21 Regulates Development of the Renal Stroma via Interaction with Wnt/ß-Catenin Signaling.

Background: Kidney formation requires coordinated interactions between multiple cell types. Input from the interstitial progenitor cells is implicated in multiple aspects of kidney development. We previously reported that transcription factor 21 (Tcf21) is required for ureteric bud branching. Here, we show that Tcf21 in Foxd1+ interstitial progenitors regulates stromal formation and differentiation via interaction with ß-catenin. Methods: We utilized the Foxd1Cre;Tcf21f/f murine kidney for morphologic analysis. We used the murine clonal mesenchymal cell lines MK3/M15 to study Tcf21 interaction with Wnt/ß-catenin. Results: Absence of Tcf21 from Foxd1+ stromal progenitors caused a decrease in stromal cell proliferation, leading to marked reduction of the medullary stromal space. Lack of Tcf21 in the Foxd1+ stromal cells also led to defective differentiation of interstitial cells to smooth-muscle cells, perivascular pericytes, and mesangial cells. Foxd1Cre;Tcf21f/f kidney showed an abnormal pattern of the renal vascular tree. The stroma of Foxd1Cre;Tcf21f/f kidney demonstrated marked reduction in ß-catenin protein expression compared with wild type. Tcf21 was bound to ß-catenin both upon ß-catenin stabilization and at basal state as demonstrated by immunoprecipitation in vitro. In MK3/M15 metanephric mesenchymal cells, Tcf21 enhanced TCF/LEF promoter activity upon ß-catenin stabilization, whereas DNA-binding deficient mutated Tcf21 did not enhance TCF/LEF promoter activity. Kidney explants of Foxd1Cre;Tcf21f/f showed low mRNA expression of stromal Wnt target genes. Treatment of the explants with CHIR, a Wnt ligand mimetic, restored Wnt target gene expression. Here, we also corroborated previous evidence that normal development of the kidney stroma is required for normal development of the Six2+ nephron progenitor cells, loop of Henle, and the collecting ducts. Conclusions: These findings suggest that stromal Tcf21 facilitates medullary stroma development by enhancing Wnt/ß-catenin signaling and promotes stromal cell proliferation and differentiation. Stromal Tcf21 is also required for the development of the adjacent nephron epithelia.

Preparation of a Single Cell Suspension from the Murine Iridocorneal Angle.

Single cell RNA sequencing is a powerful tool that can be used to identify distinct cell types and transcriptomic differences within complex tissues. It has proven to be especially useful in tissues of the eye, where investigators have identified novel cell types within the retina, anterior chamber, and iridocorneal angle and explored transcriptomic contribution to disease phenotypes in age-related macular degeneration. However, to obtain high quality results, the technique requires isolation of healthy single cells from the tissue of interest, seeking complete tissue digestion while minimizing stress and transcriptomic changes in the isolated cells prior to library preparation. Here, we present a protocol developed in our laboratory for isolation of live single cells from the murine iridocorneal angle, which includes Schlemm's canal and the trabecular meshwork, suitable for single cell RNA sequencing, flow cytometry, or other downstream analysis. Graphical abstract.

Angiopoietin-2 Inhibition of Thrombomodulin-Mediated Anticoagulation-A Novel Mechanism That May Contribute to Hypercoagulation in Critically Ill COVID-19 Patients.

Hypercoagulation and endothelial dysfunction play central roles in severe forms of COVID-19 infections, but the molecular mechanisms involved are unclear. Increased plasma levels of the inflammatory cytokine and TIE2 receptor antagonist Angiopoietin-2 were reported in severely ill COVID-19 patients. In vitro experiments suggest that Angiopoietin-2 bind and inhibits thrombomodulin. Thrombomodulin is expressed on the luminal surface of endothelial cells where it is an important member of the intrinsic anticoagulant pathway through activation of protein C. Using clinical data, mouse models, and in vitro assays, we tested if Angiopoietin-2 plays a causal role in COVID-19-associated hypercoagulation through direct inhibition of thrombin/thrombomodulin-mediated physiological anticoagulation. Angiopoietin-2 was measured in 61 patients at admission, and after 10 days in the 40 patients remaining in the ICU. We found that Angiopoietin-2 levels were increased in COVID-19 patients in correlation with disease severity, hypercoagulation, and mortality. In support of a direct effect of Angiopoietin-2 on coagulation, we found that injected Angiopoietin-2 in mice associated to thrombomodulin and resulted in a shortened tail bleeding time, decreased circulating levels of activated protein C, and increased plasma thrombin/antithrombin complexes. Conversely, bleeding time was increased in endothelial-specific Angiopoietin-2 knockout mice, while knockout of Tie2 had no effect on tail bleeding. Using in vitro assays, we found that Angiopoietin-2 inhibited thrombomodulin-mediated anticoagulation and protein C activation in human donor plasma. Our data suggest a novel in vivo mechanism for Angiopoietin-2 in COVID-19-associated hypercoagulation, implicating that Angiopoietin-2 inhibitors may be effective in the treatment of hypercoagulation in severe COVID-19 infection.

Moving from Evidence to Implementation of Breakthrough Therapies for Diabetic Kidney Disease.

Diabetic kidney disease is the most frequent cause of kidney failure, accounting for half of all cases worldwide. Moreover, deaths from diabetic kidney disease increased 106% between 1990 and 2013, with most attributed to cardiovascular disease. Recommended screening and monitoring for diabetic kidney disease are conducted in less than half of patients with diabetes. Standard-of-care treatment with an angiotensin-converting enzyme inhibitor or an angiotensin receptor blocker is correspondingly low. Sodium-glucose cotransporter 2 inhibitors, glucagon-like peptide 1 receptor agonists, and a nonsteroidal mineralocorticoid antagonist are highly effective therapies to reduce kidney and cardiovascular risks in diabetic kidney disease. However, <20% of eligible patients are receiving these agents. Critical barriers are high out-of-pocket drug costs and low reimbursement rates. Data demonstrating clinical and cost-effectiveness of diabetic kidney disease care are needed to garner payer and health care system support. The pharmaceutical industry should collaborate on value-based care by increasing access through affordable drug prices. Additionally, multidisciplinary models and communication technologies tailored to individual health care systems are needed to support optimal diabetic kidney disease care. Community outreach efforts are also central to make care accessible and equitable. Finally, it is imperative that patient preferences and priorities shape implementation strategies. Access to care and implementation of breakthrough therapies for diabetic kidney disease can save millions of lives by preventing kidney failure, cardiovascular events, and premature death. Coalitions composed of patients, families, community groups, health care professionals, health care systems, federal agencies, and payers are essential to develop collaborative models that successfully address this major public health challenge.

Endothelial Tyrosine Kinase Tie1 Is Required for Normal Schlemm's Canal Development-Brief Report.

BACKGROUND: Schlemm's canal (SC) is a large vessel residing in the iridocorneal angle and is required to regulate aqueous humor outflow. Normal SC structure and function is indispensable for maintaining normal intraocular pressure, and elevated intraocular pressure is a risk factor for development of glaucoma. Recent reports have identified a key role of the angiopoietin-Tie2 pathway for SC development and function; however, the role of the orphan receptor Tie1 has not been clarified. METHODS: We used Tie1 knock out mice to study the function of Tie1 in SC development and function. Real-time quantitative polymerase chain reaction and Western blot analyses were used to verify Tie1 deletion. High-resolution microscopy of mouse SC whole mount and cross sections were used to study SC morphology. Measurement of intraocular pressure in live mice was used to study the impact of Tie1 on SC function. RESULTS: Tie1 is highly expressed in both human and mouse SC. Tie1 knock out mice display hypomorphic SC and elevated intraocular pressure as a result of attenuated SC development. CONCLUSIONS: Tie1 is indispensable for SC development and function, supporting it as a novel target for future SC-targeted glaucoma therapies and a candidate gene for glaucoma in humans.

Cellular crosstalk regulates the aqueous humor outflow pathway and provides new targets for glaucoma therapies.

Primary congenital glaucoma (PCG) is a severe disease characterized by developmental defects in the trabecular meshwork (TM) and Schlemm's canal (SC), comprising the conventional aqueous humor outflow pathway of the eye. Recently, heterozygous loss of function variants in TEK and ANGPT1 or compound variants in TEK/SVEP1 were identified in children with PCG. Moreover, common variants in ANGPT1and SVEP1 have been identified as risk alleles for primary open angle glaucoma (POAG) in GWAS studies. Here, we show tissue-specific deletion of Angpt1 or Svep1 from the TM causes PCG in mice with severe defects in the adjacent SC. Single-cell transcriptomic analysis of normal and glaucomatous Angpt1 deficient eyes allowed us to identify distinct TM and SC cell populations and discover additional TM-SC signaling pathways. Furthermore, confirming the importance of angiopoietin signaling in SC, delivery of a recombinant ANGPT1-mimetic promotes developmental SC expansion in healthy and Angpt1 deficient eyes, blunts intraocular pressure (IOP) elevation and RGC loss in a mouse model of PCG and lowers IOP in healthy adult mice. Our data highlight the central role of ANGPT1-TEK signaling and TM-SC crosstalk in IOP homeostasis and provide new candidates for SC-targeted glaucoma therapy.