Lung injury-induced activated endothelial cell states persist in aging-associated progressive fibrosis.

Progressive lung fibrosis is associated with poorly understood aging-related endothelial cell dysfunction. To gain insight into endothelial cell alterations in lung fibrosis we performed single cell RNA-sequencing of bleomycin-injured lungs from young and aged mice. Analysis reveals activated cell states enriched for hypoxia, glycolysis and YAP/TAZ activity in ACKR1+ venous and TrkB+ capillary endothelial cells. Endothelial cell activation is prevalent in lungs of aged mice and can also be detected in human fibrotic lungs. Longitudinal single cell RNA-sequencing combined with lineage tracing demonstrate that endothelial activation resolves in young mouse lungs but persists in aged ones, indicating a failure of the aged vasculature to return to quiescence. Genes associated with activated lung endothelial cells states in vivo can be induced in vitro by activating YAP/TAZ. YAP/TAZ also cooperate with BDNF, a TrkB ligand that is reduced in fibrotic lungs, to promote capillary morphogenesis. These findings offer insights into aging-related lung endothelial cell dysfunction that may contribute to defective lung injury repair and persistent fibrosis.

Kidney Disease and Viral Infection in COVID-19: Why Are Kidney Organoid and Biopsy Studies Not in Agreement?

CONTEXT: The clinical course of coronavirus disease-19 (COVID-19) can be complicated by acute kidney injury and proteinuria. Kidney cells express receptors for SARS-CoV-2, the virus responsible for COVID-19. Direct infection of the kidney parenchyma by SARS-CoV-2 has been proposed as the cause of renal dysfunction in COVID-19. Subject of Review: Kidney organoids derived from human embryonic stem cells or induced pluripotent cells can be reproducibly infected by SARS-CoV-2 in vitro and used to study therapeutics. However, kidney biopsy studies of COVID-19 patients with renal dysfunction have shown no evidence of viral infection. Second Opinion: Kidney organoids are susceptible to SARS-CoV-2 infection, which is probably facilitated by their limited architectural complexity and maturation compared to the intact organ and by the in vitro culture conditions. Conversely, kidneys in COVID-19 patients appear resistant to infection and may be injured through indirect mechanisms mediated by the host response to the respiratory viral infection, genetic susceptibility to the immune response, physiological disturbances, and therapies. More studies are needed to better understand why kidney organoids are more susceptible than mature kidneys to SARS-CoV-2 infection and further characterize the mechanisms of kidney injury in COVID-19.

Dysfunctional ERG signaling drives pulmonary vascular aging and persistent fibrosis.

Vascular dysfunction is a hallmark of chronic diseases in elderly. The contribution of the vasculature to lung repair and fibrosis is not fully understood. Here, we performed an epigenetic and transcriptional analysis of lung endothelial cells (ECs) from young and aged mice during the resolution or progression of bleomycin-induced lung fibrosis. We identified the transcription factor ETS-related gene (ERG) as putative orchestrator of lung capillary homeostasis and repair, and whose function is dysregulated in aging. ERG dysregulation is associated with reduced chromatin accessibility and maladaptive transcriptional responses to injury. Loss of endothelial ERG enhances paracrine fibroblast activation in vitro, and impairs lung fibrosis resolution in young mice in vivo. scRNA-seq of ERG deficient mouse lungs reveales transcriptional and fibrogenic abnormalities resembling those associated with aging and human lung fibrosis, including reduced number of general capillary (gCap) ECs. Our findings demonstrate that lung endothelial chromatin remodeling deteriorates with aging leading to abnormal transcription, vascular dysrepair, and persistent fibrosis following injury.

Digital spatial profiling of collapsing glomerulopathy.

Collapsing glomerulopathy is a histologically distinct variant of focal and segmental glomerulosclerosis that presents with heavy proteinuria and portends a poor prognosis. Collapsing glomerulopathy can be triggered by viral infections such as HIV or SARS-CoV-2. Transcriptional profiling of collapsing glomerulopathy lesions is difficult since only a few glomeruli may exhibit this histology within a kidney biopsy and the mechanisms driving this heterogeneity are unknown. Therefore, we used recently developed digital spatial profiling (DSP) technology which permits quantification of mRNA at the level of individual glomeruli. Using DSP, we profiled 1,852 transcripts in glomeruli isolated from formalin fixed paraffin embedded sections from HIV or SARS-CoV-2-infected patients with biopsy-confirmed collapsing glomerulopathy and used normal biopsy sections as controls. Even though glomeruli with collapsing features appeared histologically similar across both groups of patients by light microscopy, the increased resolution of DSP uncovered intra- and inter-patient heterogeneity in glomerular transcriptional profiles that were missed in early laser capture microdissection studies of pooled glomeruli. Focused validation using immunohistochemistry and RNA in situ hybridization showed good concordance with DSP results. Thus, DSP represents a powerful method to dissect transcriptional programs of pathologically discernible kidney lesions.

A Diverse Spectrum of Immune Complex- and Complement-Mediated Kidney Diseases Is Associated With Mantle Cell Lymphoma.

Introduction: There are limited reports on kidney biopsy findings in patients with mantle cell lymphoma (MCL). Methods: We initiated a multi-institutional, retrospective review of kidney biopsy findings in patients with active and treated MCL. Results: A total of 30 patients with MCL and kidney biopsies were identified, with a median age of 67 (range 48-87) years, 73% of whom were men. A total of 20 patients had active MCL at the time of biopsy, of whom 14 (70%) presented with acute kidney injury (AKI), proteinuria and/or hematuria, and biopsy findings potentially attributable to lymphoma. Of the 14, 11 had immune complex (IC) or complement-mediated (C3) disease including proliferative glomerulonephritis (GN) with monotypic Ig deposits (PGNMID [2]), C3GN, (2), secondary membranous nephropathy (MN [3]), tubular basement membrane (TBM) deposits (2), and modest lupus-like GN (2). Lymphomatous infiltration was present in 8 of the 20 patients, 5 with coincident IC or C3 lesions. A total of 6 patients with available follow-up were treated for MCL, all with clinical remission of GN (2 PGNMID, 2 C3GN, and 2 MN). Conclusion: MCL is associated with diverse monoclonal and polyclonal glomerular and extra-glomerular IC and C3 disease. For patients with active MCL and kidney dysfunction requiring biopsy, 70% had findings due or potentially due to lymphoma, including 55% with IC or C3 disease and 40% had lymphomatous kidney infiltration. IC and C3GN in the setting of active MCL was responsive to lymphoma-directed therapy.

Early Transplant Arteriopathy in Kidney Transplantation.

BACKGROUND: Early dysfunction of renal allografts may be associated with vascular injury, which raises the specter of active rejection processes that require medical intervention. In our practice, we have encountered patients who present with delayed graft function and demonstrate a unique pattern of endothelial cell injury that raises concern for rejection in their biopsy. Therefore, we sought to systematically determine the biopsy characteristics and outcome of these patients. METHODS: During a 17-year period at the University of Washington in Seattle, United States, we identified 24 cases of a distinct arterial vasculopathy presenting in the first year posttransplantation. This early transplant arteriopathy (ETA) is characterized by endothelial cell swelling and intimal edema but without the intimal arteritis that defines vascular rejection. RESULTS: Approximately 1% of transplant biopsies during the study period showed ETA, almost all of which were in deceased donor organs (96%), and most presented with delayed graft function (54%) or increased serum creatinine (38%) soon after transplantation (median 13 days; range, 5-139). In this study, 77% of patients were managed expectantly, with only 2 patients (7.6%) subsequently developing acute vascular rejection. Except for 1 patient who died, all patients had functioning allografts at 1 year follow-up. CONCLUSION: Recognizing ETA and distinguishing it from vascular rejection is important to prevent over-treatment because most patients appear to recover allograft function rapidly with expectant management.

Multicenter Clinicopathologic Correlation of Kidney Biopsies Performed in COVID-19 Patients Presenting With Acute Kidney Injury or Proteinuria.

RATIONALE & OBJECTIVE: Kidney biopsy data inform us about pathologic processes associated with infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We conducted a multicenter evaluation of kidney biopsy findings in living patients to identify various kidney disease pathology findings in patients with coronavirus disease 2019 (COVID-19) and their association with SARS-CoV-2 infection. STUDY DESIGN: Case series. SETTING & PARTICIPANTS: We identified 14 native and 3 transplant kidney biopsies performed for cause in patients with documented recent or concurrent SARS-CoV-2 infection treated at 7 large hospital systems in the United States. OBSERVATIONS: Men and women were equally represented in this case series, with a higher proportion of Black (n=8) and Hispanic (n=5) patients. All 17 patients had SARS-CoV-2 infection confirmed by reverse transcriptase-polymerase chain reaction, but only 3 presented with severe COVID-19 symptoms. Acute kidney injury (n=15) and proteinuria (n=11) were the most common indications for biopsy and these symptoms developed concurrently or within 1 week of COVID-19 symptoms in all patients. Acute tubular injury (n=14), collapsing glomerulopathy (n=7), and endothelial injury/thrombotic microangiopathy (n=6) were the most common histologic findings. 2 of the 3 transplant recipients developed active antibody-mediated rejection weeks after COVID-19. 8 patients required dialysis, but others improved with conservative management. LIMITATIONS: Small study size and short clinical follow-up. CONCLUSIONS: Cases of even symptomatically mild COVID-19 were accompanied by acute kidney injury and/or heavy proteinuria that prompted a diagnostic kidney biopsy. Although acute tubular injury was seen among most of them, uncommon pathology such as collapsing glomerulopathy and acute endothelial injury were detected, and most of these patients progressed to irreversible kidney injury and dialysis.

Fibrillary Glomerulonephritis: Clinicopathologic Features and Atypical Cases from a Multi-Institutional Cohort.

BACKGROUND AND OBJECTIVES: Fibrillary GN has been defined as an immune complex-mediated GN with amyloid-like fibrils larger than amyloid which are IgG positive and Congo red negative. With discovery of DNAJB9 as a highly sensitive and specific marker for fibrillary GN, the specificity of the morphologic criteria for establishing the diagnosis of fibrillary GN has come into question. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We sought to (1) determine anatomic characteristics that best define fibrillary GN and (2) identify clinical and pathologic features that predict outcomes. RESULTS: We retrospectively reviewed kidney biopsies from patients diagnosed with fibrillary GN or suspected fibrillary GN between 1997 and 2017 (n=266, 65% female, median age 61). Approximately 11% of kidney biopsies had one or more unusual feature including monotypic deposits, Congo red positivity, or unusual fibril diameter. Fibrillary GN as a possible monoclonal gammopathy of renal significance represented <1% of cases. Immunostaining for DNAJB9 confirmed fibrillary GN in 100% of cases diagnosed as fibrillary GN and 79% of atypical cases diagnosed as possible fibrillary GN. At a median time of 24 months (interquartile range, 8-46 months) after biopsy (n=100), 53% of patients reached the combined primary outcome of ESKD or death, 18% had CKD, and 18% had partial remission. On multivariable analysis, male sex (adjusted hazard ratio [aHR], 3.82; 95% confidence interval [95% CI], 1.97 to 7.37) and eGFR were the most significant predictors of primary outcome (aHR of 8.02 if eGFR <30 ml/min per 1.73 m2 [95% CI, 1.85 to 34.75]; aHR of 6.44 if eGFR 30 to <45 ml/min per 1.73 m2 [95% CI, 1.38 to 29.99]). Immunosuppressive therapy with rituximab was significantly associated with stabilization of disease progression. CONCLUSIONS: Detection of DNAJB9 is a useful diagnostic tool for diagnosing atypical forms of fibrillary GN. The outcomes for fibrillary GN are poor and progression to ESKD is influenced predominantly by the degree of kidney insufficiency at the time of diagnosis and male sex. Rituximab may help preserve kidney function for select patients with fibrillary GN. PODCAST: This article contains a podcast at https://www.asn-online.org/media/podcast/CJASN/2019_11_04_CJN03870319.mp3.

The plaque-aortic ring assay: a new method to study human atherosclerosis-induced angiogenesis.

Progression of atherosclerotic plaques into life-threatening lesions is associated with angiogenesis which contributes to intraplaque hemorrhages and plaque instability. The lack of adequate models for the study of human plaque-induced angiogenesis has limited progress in this field. We describe here a novel ex vivo model which fills this gap. Plaques obtained from 15 patients who underwent endarterectomy procedures were co-cultured in collagen gels with rat aorta rings which served as read-out of human plaque angiogenic activity. The majority of plaque fragments markedly stimulated angiogenic sprouting from the aortic rings while concurrently promoting the outgrowth of resident macrophages from the aortic adventitia. This stimulatory activity correlated with the presence of intraplaque macrophages. Proteomic analysis of plaque secretomes revealed heterogeneity of macrophage-stimulatory cytokine and angiogenic factor production by different plaques. VEGF was identified in some of the plaque secretomes. Antibody-mediated blockade of VEGF had significant but transient inhibitory effect on angiogenesis, which suggested redundancy of plaque-derived angiogenic stimuli. Pharmacologic ablation of adventitial macrophages permanently impaired the angiogenic response of aortic rings to plaque stimuli. Our results show that human plaque-induced angiogenesis can be reproduced ex vivo using rat aortic rings as read-out of plaque angiogenic activity. This model can be used to identify key cellular and molecular mechanisms responsible for the neovascularization of human plaques.

IgA-dominant glomerulonephritis with a membranoproliferative pattern of injury.

Immunoglobulin A (IgA)-dominant membranoproliferative glomerulonephritis (MPGN) is a descriptive term for renal biopsies in which differential diagnoses of unusual IgA nephropathy (IgAN), infection-related GN, or other etiologies are considered. We sought to understand clinical and pathologic features of this finding. Native kidney biopsies with IgA-dominant immune deposits and diffuse MPGN features without significant exudative features or subepithelial deposits were retrospectively reviewed. Two groups (n = 27, 33 biopsies) were identified: patients with chronic liver disease and those without. Patients without chronic liver disease (n = 15) were men (73%, age 40) who presented with nephrotic-range proteinuria, hematuria, renal insufficiency, negative serologic studies, and no history of infection. At a median interval of 3 years, 11 had available follow-up information. Three (27%) progressed to end-stage renal disease. One had recurrent IgA-dominant GN in the renal allograft less than 1 year posttransplant. Four of 5 patients with repeat biopsies had persistent IgA-dominant MPGN. Patients with chronic liver disease (n = 12) had similar biopsy findings, but 42% had concurrent infections, some occult. At a median interval of 7 weeks, 8 patients (80% of those with follow-up) had died and 2 were dialysis dependent. In conclusion, IgA-dominant MPGN was seen in 2 clinical cohorts in this study. In patients without chronic liver disease, this appears to represent either a unique clinicopathologic entity with a poorer prognosis than IgAN or an aggressive variant of IgAN. Patients with chronic liver disease often have underlying infection, and regardless of treatment, die within 1 year because of complex medical conditions.

Hyperactive FOXO1 results in lack of tip stalk identity and deficient microvascular regeneration during kidney injury.

Loss of the microvascular (MV) network results in tissue ischemia, loss of tissue function, and is a hallmark of chronic diseases. The incorporation of a functional vascular network with that of the host remains a challenge to utilizing engineered tissues in clinically relevant therapies. We showed that vascular-bed-specific endothelial cells (ECs) exhibit differing angiogenic capacities, with kidney microvascular endothelial cells (MVECs) being the most deficient, and sought to explore the underlying mechanism. Constitutive activation of the phosphatase PTEN in kidney MVECs resulted in impaired PI3K/AKT activity in response to vascular endothelial growth factor (VEGF). Suppression of PTEN in vivo resulted in microvascular regeneration, but was insufficient to improve tissue function. Promoter analysis of the differentially regulated genes in KMVECs suggests that the transcription factor FOXO1 is highly active and RNAseq analysis revealed that hyperactive FOXO1 inhibits VEGF-Notch-dependent tip-cell formation by direct and indirect inhibition of DLL4 expression in response to VEGF. Inhibition of FOXO1 enhanced angiogenesis in human bio-engineered capillaries, and resulted in microvascular regeneration and improved function in mouse models of injury-repair.

The Aortic Ring Assay and Its Use for the Study of Tumor Angiogenesis.

This chapter describes protocols developed in our laboratory to prepare and analyze angiogenic cultures of rat aorta. Rings of rat aorta cultured in collagen gels produce neovessel outgrowths which reproduce ex vivo key steps of the angiogenic process including endothelial migration, proliferation, proteolytic digestion of the extracellular matrix, capillary tube formation, pericyte recruitment, and vascular regression. The angiogenic response of aortic explants can be stimulated with growth factors or inhibited with anti-angiogenic molecules. Aortic ring cultures can also be used to study tumor angiogenesis. Protocols outlined in this chapter describe how this assay can be modified to investigate the angiogenic activity of cancer cells.

Hypoxia paradoxically inhibits the angiogenic response of isolated vessel explants while inducing overexpression of vascular endothelial growth factor.

This study was designed to investigate how changes in O2 levels affected angiogenesis in vascular organ culture. Although hypoxia is a potent inducer of angiogenesis, aortic rings cultured in collagen paradoxically failed to produce an angiogenic response in 1-4 % O2. Additionally, aortic neovessels preformed in atmospheric O2 lost pericytes and regressed at a faster rate than control when exposed to hypoxia. Aortic explants remained viable in hypoxia and produced an angiogenic response when returned to atmospheric O2. Hypoxic aortic rings were unresponsive to VEGF, while increased oxygenation of the system dose-dependently enhanced VEGF-induced angiogenesis. Hypoxia-induced refractoriness to angiogenic stimulation was not restricted to the aorta because similar results were obtained with vena cava explants or isolated endothelial cells. Unlike endothelial cells, aorta-derived mural cells were unaffected by hypoxia. Hypoxia downregulated expression in aortic explants of key signaling molecules including VEGFR2, NRP1 and Prkc-beta while upregulating expression of VEGFR1. Medium conditioned by hypoxic cultures exhibited angiostatic and anti-VEGF activities likely mediated by sVEGFr1. Hypoxia reduced expression of VEGFR1 and VEGFR2 in endothelial cells while upregulating VEGFR1 in macrophages and VEGF in both macrophages and mural cells. Thus, changes in O2 levels profoundly affect the endothelial response to angiogenic stimuli. These results suggest that hypoxia-induced angiogenesis is fine-tuned by complex regulatory mechanisms involving not only production of angiogenic factors including VEGF but also differential regulation of VEGFR expression in different cell types and production of inhibitors of VEGF function such as sVEGFR1.

Renal ApoA-1 amyloidosis with Glu34Lys mutation and intra-amyloid lipid accumulation.

Apolipoprotein A-1 (ApoA-1) amyloidosis occurs as a nonhereditary condition in atherosclerotic plaques, but it can also manifest as a hereditary disorder caused by mutations of the APOA1 gene. Hereditary ApoA-1 amyloidosis presents with diverse organ involvement based on the position of the mutation. We describe a case of ApoA-1 amyloidosis with a Glu34Lys mutation; testicular, conjunctival, and renal involvement; and the notable finding of lipid deposition within the amyloid deposits.

Combined crystalline podocytopathy and tubulopathy associated with multiple myeloma.

Lymphoplasmacytic neoplasms cause a wide range of injuries to the kidney exemplified by light chain cast nephropathy and amyloidosis. Filtered paraproteins can also accumulate within kidney cells and cause direct cytotoxic injury. Rarely, paraproteins that are resistant to proteolysis can crystallize within proximal tubules and cause acute tubular injury. In contrast, accumulation of crystallized paraproteins in other kidney cells, especially podocytes, is exceptional. Here, we report the finding of crystalline inclusions within podocytes and proximal tubules in a patient who presented with a combined nephrotic syndrome and Fanconi syndrome. Further workup revealed previously unsuspected multiple myeloma and elevated serum free light chains, highlighting the protean presentation of paraprotein-mediated injuries to the kidney.

Regulation of angiogenesis, mural cell recruitment and adventitial macrophage behavior by Toll-like receptors.

The angiogenic response to injury can be studied by culturing rat or mouse aortic explants in collagen gels. Gene expression studies show that aortic angiogenesis is preceded by an immune reaction with overexpression of Toll-like receptors (TLRs) and TLR-inducible genes. TLR1, 3, and 6 are transiently upregulated at 24 h whereas TLR2, 4, and 8 expression peaks at 24 h but remains elevated during angiogenesis and vascular regression. Expression of TLR5, 7 and 9 steadily increases over time and is highest during vascular regression. Studies with isolated cells show that TLRs are expressed at higher levels in aortic macrophages compared to endothelial or mural cells with the exception of TLR2 and TLR9 which are more abundant in the aortic endothelium. LPS and other TLR ligands dose dependently stimulate angiogenesis and vascular endothelial growth factor production. TLR9 ligands also influence the behavior of nonendothelial cell types by blocking mural cell recruitment and inducing formation of multinucleated giant cells by macrophages. TLR9-induced mural cell depletion is associated with reduced expression of the mural cell recruiting factor PDGFB. The spontaneous angiogenic response of the aortic rings to injury is reduced in cultures from mice deficient in myeloid differentiation primary response 88 (MyD88), a key adapter molecule of TLRs, and following treatment with an inhibitor of the NFκB pathway. These results suggest that the TLR system participates in the angiogenic response of the vessel wall to injury and may play an important role in the regulation of inflammatory angiogenesis in reactive and pathologic processes.

SCNH2 is a novel apelinergic family member acting as a potent mitogenic and chemotactic factor for both endothelial and epithelial cells.

The gut hormone apelin is a major therapeutic focus for several diseases involving inflammation and aberrant cell growth. We investigated whether apelin-36 contained alternative bioactive peptides associated with normal physiology or disease. Amino acid sequence analysis of apelin-36 identified an amidation motif consistent with the formation of a secondary bioactive peptide (SCNH2). SCNH2 is proven to be mitogenic and chemotactic in normal/malignant cells and augments angiogenesis via a PTX-resistant/CT-X-sensitive G protein-coupled receptor (GPCR). Notably, SCNH2 is substantially more potent and sensitive than apelin-13 and vascular endothelial growth factor-A. Endogenous SCNH2 is highly expressed in human tumors and placenta and in mouse embryonic tissues. Our findings demonstrate that SCNH2 is a new apelinergic member with critical pluripotent roles in angiogenesis related diseases and embryogenesis via a non-APJ GPCR.

Diffuse proliferative glomerulonephritis associated with cetuximab, an epidermal growth factor receptor inhibitor.

Cetuximab is an epidermal growth factor receptor inhibitor used for advanced squamous cell carcinoma of the head and neck. We report an unusual case of diffuse proliferative and crescentic glomerulonephritis in a 67-year-old man in close temporal association with cetuximab treatment for recurrent oral squamous cell carcinoma. The patient presented with acute renal failure and nephrotic-range proteinuria. Kidney biopsy showed diffuse proliferative and focally crescentic glomerulonephritis associated with immunoglobulin A (IgA)-dominant immune-complex deposition within glomerular capillary walls and mesangium. The patient showed dramatic improvement in kidney function after discontinuation of cetuximab therapy and a short course of cyclophosphamide and steroid. The clinical outcome of this case suggests that cetuximab therapy may trigger or exacerbate IgA-mediated glomerular injury and warrants close monitoring of kidney function in patients treated with this epidermal growth factor receptor inhibitor.

Macrophage Wnt-Calcineurin-Flt1 signaling regulates mouse wound angiogenesis and repair.

The treatment of festering wounds is one of the most important aspects of medical care. Macrophages are important components of wound repair, both in fending off infection and in coordinating tissue repair. Here we show that macrophages use a Wnt-Calcineurin-Flt1 signaling pathway to suppress wound vasculature and delay repair. Conditional mutants deficient in both Wntless/GPR177, the secretory transporter of Wnt ligands, and CNB1, the essential component of the nuclear factor of activated T cells dephosporylation complex, displayed enhanced angiogenesis and accelerated repair. Furthermore, in myeloid-like cells, we show that noncanonical Wnt activates Flt1, a naturally occurring inhibitor of vascular endothelial growth factor-A-mediated angiogenesis, but only when calcineurin function is intact. Then, as expected, conditional deletion of Flt1 in macrophages resulted in enhanced wound angiogenesis and repair. These results are consistent with the published link between enhanced angiogenesis and enhanced repair, and establish novel therapeutic approaches for treatment of wounds.

The acute phase reactant orosomucoid-1 is a bimodal regulator of angiogenesis with time- and context-dependent inhibitory and stimulatory properties.

BACKGROUND: Tissues respond to injury by releasing acute phase reaction (APR) proteins which regulate inflammation and angiogenesis. Among the genes upregulated in wounded tissues are tumor necrosis factor-alpha (TNFα) and the acute phase reactant orosomucoid-1 (ORM1). ORM1 has been shown to modulate the response of immune cells to TNFα, but its role on injury- and TNFα-induced angiogenesis has not been investigated. This study was designed to characterize the role of ORM1 in the angiogenic response to injury and TNFα. METHODS AND RESULTS: Angiogenesis was studied with in vitro, ex vivo, and in vivo angiogenesis assays. Injured rat aortic rings cultured in collagen gels produced an angiogenic response driven by macrophage-derived TNFα. Microarray analysis and qRT-PCR showed that TNFα and ORM1 were upregulated prior to angiogenic sprouting. Exogenous ORM1 delayed the angiogenic response to injury and inhibited the proangiogenic effect of TNFα in cultures of aortic rings or isolated endothelial cells, but stimulated aortic angiogenesis over time while promoting VEGF production and activity. ORM1 inhibited injury- and TNFα-induced phosphorylation of MEK1/2 and p38 MAPK in aortic rings, but not of NFκB. This effect was injury/TNFα-specific since ORM1 did not inhibit VEGF-induced signaling, and cell-specific since ORM1 inhibited TNFα-induced phosphorylation of MEK1/2 and p38 MAPK in macrophages and endothelial cells, but not mural cells. Experiments with specific inhibitors demonstrated that the MEK/ERK pathway was required for angiogenesis. ORM1 inhibited angiogenesis in a subcutaneous in vivo assay of aortic ring-induced angiogenesis, but stimulated developmental angiogenesis in the chorioallantoic membrane (CAM) assay. CONCLUSION: ORM1 regulates injury-induced angiogenesis in a time- and context-dependent manner by sequentially dampening the initial TNFα-induced angiogenic response and promoting the downstream stimulation of the angiogenic process by VEGF. The context-dependent nature of ORM1 angioregulatory function is further demonstrated in the CAM assay where ORM1 stimulates developmental angiogenesis without exerting any inhibitory activity.