Epidermal growth factor deficiency predisposes to progressive renal disease.

Epidermal growth factor (EGF) is produced in the kidney by thick ascending limbs of the loop of Henle and by distal convoluted tubules (DCTs). Reduced urinary EGF levels have been associated with chronic kidney disease but it is not known whether physiological levels of EGF protect the kidney from progressive renal disease. Here, we show that EGF-deficient mice on a mixed genetic background had increased urinary microalbumin, and a subset of these mice developed severe progressive renal disease with azotemia that was not seen in WT or TGFα-deficient littermates with this mixed genetic background. These azotemic EGF-deficient mice developed crescentic glomerulonephritis linked to HB-EGF/EGFR hyperactivation in glomeruli, as well as attenuation of the proximal tubule brush border, distal convoluted tubule (DCT) dilatation, and kidney fibrosis associated with renal ß-catenin/mTOR hyperactivation. The observation of these severe renal pathologies only in a subset of EGF-deficient mice suggests that independent segregation of strain-specific modifier alleles contributes to the severity of the renal abnormalities that only manifest when EGF is lacking. These findings link the lack of EGF to renal pathologies in the adult mammalian kidney, in support of a role of physiological levels of EGF for maintaining the function of glomeruli, proximal tubules, and DCTs. These observations suggest that diminished EGF levels predispose kidneys to progressive renal disease.

Choriocapillaris Degeneration in Geographic Atrophy.

Early age-related macular degeneration (AMD) is characterized by degeneration of the choriocapillaris, the vascular supply of retinal photoreceptor cells. We assessed vascular loss during disease progression in the choriocapillaris and larger vessels in the deeper choroid. Human donor maculae from controls (n = 99), early AMD (n = 35), or clinically diagnosed with geographic atrophy (GA; n = 9, collected from outside the zone of retinal pigment epithelium degeneration) were evaluated using Ulex europaeus agglutinin-I labeling to discriminate between vessels with intact endothelial cells and ghost vessels. Morphometric analyses of choriocapillaris density (cross-sectional area of capillary lumens divided by length) and of vascular lumen/stroma ratio in the outer choroid were performed. Choriocapillaris loss was observed in early AMD (Bonferroni-corrected P = 0.024) with greater loss in GA (Bonferroni-corrected P < 10-9), even in areas of intact retinal pigment epithelium. In contrast, changes in lumen/stroma ratio in the outer choroid were not found to differ between controls and AMD or GA eyes (P > 0.05), suggesting choriocapillaris changes are more prevalent in AMD than those in the outer choroid. In addition, vascular endothelial growth factor-A levels were negatively correlated with choriocapillaris vascular density. These findings support the concept that choroidal vascular degeneration, predominantly in the microvasculature, contributes to dry AMD progression. Addressing capillary loss in AMD remains an important translational target.

Effects of chronically increased VEGF-A on the aging heart.

Whether approaches to chronically increase VEGF-A in the heart may have beneficial effects and prevent the development of heart failure, in part by improving cardiac perfusion, or whether this increase could have detrimental effects on cardiac performance in the aging heart, has not been tested yet. In this study, a genetic mouse model with a chronic increase in VEGF-A in the heart is shown to have increased cardiac angiogenesis and develop cardiac hypertrophy with enhanced basal cardiac performance with age progression. However, in aged hearts, this increase in VEGF-A was associated with higher expression of fetal cardiac genes and reduced cardiac performance after ß-agonistic stress, features consistent with pathologic cardiac hypertrophy. Expression of Nod-like receptor protein (NLRP)-3 was increased in the hearts of the mice, and its genetic inactivation prevented increased fetal cardiac gene expression and partially rescued the impaired cardiac performance after ß-agonistic stimulation in aged hearts without reducing cardiac angiogenesis or hypertrophy. Thus, although a chronic increase in cardiac VEGF-A may improve cardiac perfusion, long-term upregulation of VEGF-A leads to reduced cardiac performance under stress, an effect that can be partially inhibited by NLRP3 inactivation. Targeting NLRP3 shifts the VEGF-A-induced cardiac hypertrophy from a pathologic toward a more physiologic hypertrophy.-Marneros, A. G. Effects of chronically increased VEGF-A on the aging heart.

Targeting Platelet-Derived Growth Factor Receptor ß(+) Scaffold Formation Inhibits Choroidal Neovascularization.

Neovascular age-related macular degeneration is among the most common causes of irreversible blindness and manifests with choroidal neovascularization (CNV). Anti-vascular endothelial growth factor-A therapies are only partially effective and their chronic administration may impair functions of the choriocapillaris and retina. Thus, novel therapeutic targets are needed urgently. We have observed in a laser-induced model of CNV that a platelet-derived growth factor receptor ß positive (PDGFRß(+)) scaffold is formed before infiltration of neovessels into this scaffold to form CNV lesions, and that this scaffold limits the extent of neovascularization. Based on these observations we hypothesized that ablation of proliferating PDGFRß(+) cells to prevent the formation of this scaffold might inhibit CNV growth and present a novel therapeutic approach for neovascular age-related macular degeneration. To test this hypothesis we targeted proliferating PDGFRß(+) cells through independent distinct approaches after laser injury: i) by using an inducible genetic model to inhibit specifically proliferating PDGFRß(+) cells, ii) by treating mice with a neutralizing anti-PDGFRß antibody, iii) by administering an anti-PDGF-AB/BB aptamer, and iv) by using small chemical inhibitor approaches. The results show that therapeutic targeting of proliferating PDGFRß(+) cells potently inhibits the formation of the pericyte-like scaffold, with concomitant attenuation of CNV. Moreover, we show that early inhibition of PDGFRß(+) cell proliferation before neovessel formation is sufficient to inhibit scaffold formation and neovascularization.

Mutations in KCTD1 cause scalp-ear-nipple syndrome.

Scalp-ear-nipple (SEN) syndrome is a rare, autosomal-dominant disorder characterized by cutis aplasia of the scalp; minor anomalies of the external ears, digits, and nails; and malformations of the breast. We used linkage analysis and exome sequencing of a multiplex family affected by SEN syndrome to identify potassium-channel tetramerization-domain-containing 1 (KCTD1) mutations that cause SEN syndrome. Evaluation of a total of ten families affected by SEN syndrome revealed KCTD1 missense mutations in each family tested. All of the mutations occurred in a KCTD1 region encoding a highly conserved bric-a-brac, tram track, and broad complex (BTB) domain that is required for transcriptional repressor activity. KCTD1 inhibits the transactivation of the transcription factor AP-2α (TFAP2A) via its BTB domain, and mutations in TFAP2A cause cutis aplasia in individuals with branchiooculofacial syndrome (BOFS), suggesting a potential overlap in the pathogenesis of SEN syndrome and BOFS. The identification of KCTD1 mutations in SEN syndrome reveals a role for this BTB-domain-containing transcriptional repressor during ectodermal development.

VEGF-A and the NLRP3 Inflammasome in Age-Related Macular Degeneration.

The pathomechanisms that lead to age-related macular degeneration (AMD) are only partially understood. The NLRP3 inflammasome has been shown to be activated in the retinal pigment epithelium (RPE) in eyes with AMD. However, it is not known whether inflammasome activation is a cause or consequence of pathologic changes in AMD. A roadblock to defining the role of inflammasome activation and pathways that regulate it for AMD has been the lack of a mouse model that forms AMD-like pathologies in an age-dependent manner in which the role of the inflammasome can be investigated using genetic studies. We have recently identified such a mouse model, in which increased VEGF-A levels result in early degenerative changes of the RPE, followed by cardinal features of both nonexudative and neovascular AMD. Importantly, higher VEGF-A levels lead to increased oxidative damage and a sub-retinal inflammatory infiltrate that are associated with NLRP3 inflammasome activation in the RPE. Targeting the NLRP3 inflammasome inhibited AMD-like pathologies in these mice. These findings suggest that inhibiting the NLRP3 inflammasome or pathways that regulate it may provide novel therapeutic approaches for the treatment of both forms of AMD.

BMS1 is mutated in aplasia cutis congenita.

Aplasia cutis congenita (ACC) manifests with localized skin defects at birth of unknown cause, mostly affecting the scalp vertex. Here, genome-wide linkage analysis and exome sequencing was used to identify the causative mutation in autosomal dominant ACC. A heterozygous Arg-to-His missense mutation (p.R930H) in the ribosomal GTPase BMS1 is identified in ACC that is associated with a delay in 18S rRNA maturation, consistent with a role of BMS1 in processing of pre-rRNAs of the small ribosomal subunit. Mutations that affect ribosomal function can result in a cell cycle defect and ACC skin fibroblasts with the BMS1 p.R930H mutation show a reduced cell proliferation rate due to a p21-mediated G1/S phase transition delay. Unbiased comparative global transcript and proteomic analyses of ACC fibroblasts with this mutation confirm a central role of increased p21 levels for the ACC phenotype, which are associated with downregulation of heterogenous nuclear ribonucleoproteins (hnRNPs) and serine/arginine-rich splicing factors (SRSFs). Functional enrichment analysis of the proteomic data confirmed a defect in RNA post-transcriptional modification as the top-ranked cellular process altered in ACC fibroblasts. The data provide a novel link between BMS1, the cell cycle, and skin morphogenesis.

Doxycycline inhibits polarization of macrophages to the proangiogenic M2-type and subsequent neovascularization.

Macrophages occur along a continuum of functional states between M1-type polarized macrophages with antiangiogenic and antitumor activity and M2-type polarized macrophages, which have been implicated to promote angiogenesis and tumor growth. Proangiogenic M2-type macrophages promote various pathologic conditions, including choroidal neovascularization in models of neovascular age-related macular degeneration, or certain cancers, such as glioblastoma multiforme. Thus, a potential novel therapeutic approach to target pathological angiogenesis in these conditions would be to inhibit the polarization of macrophages toward the proangiogenic M2-type. However, no pharmacological inhibitors of M2-type macrophage polarization have been identified yet. Here we performed an unbiased pharmacological and small chemical screen to identify drugs that inhibit proangiogenic M2-type macrophage polarization and block pathologic macrophage-driven neovascularization. We identified the well tolerated and commonly used antibiotic doxycycline as a potent inhibitor of M2-type polarization of macrophages. Doxycycline inhibited, in a dose-dependent manner, M2-type polarization of human and bone marrow-derived mouse macrophages without affecting cell viability. Furthermore, doxycycline inhibited M2-type macrophage polarization and subsequent neovascularization in vivo in a laser injury model of choroidal neovascularization. Thus, doxycycline could be used to enhance current antiangiogenic treatment approaches in various conditions that are promoted by proangiogenic M2-type macrophages, including neovascular age-related macular degeneration and certain cancers.

Age- and gene-dosage-dependent cre-induced abnormalities in the retinal pigment epithelium.

To conditionally inactivate genes in the retinal pigment epithelium (RPE) transgenic mouse strains have been developed, in which Cre recombinase (Cre) expression is driven by an RPE-specific gene promoter. The RPE is a quiescent epithelium, and continuous expression of Cre could affect its function. Here, we tested the hypothesis that continuous postnatal Cre expression in the RPE may lead to cellular abnormalities, which may depend on both age and Cre gene dosage. We therefore examined the eyes of homozygous and heterozygous VMD2-Cre mice at various ages. In VMD2-Cre heterozygous mice variable progressive age-dependent RPE abnormalities were noticed, including attenuation of phalloidin and cytoplasmic active ß-catenin staining, reduced cell size, and loss of the typical honeycomb pattern of RPE morphology in those RPE cells that stained for Cre. These morphological RPE abnormalities were not noticed in Cre-negative RPE cells in VMD2-Cre or age-matched control mice. In addition, an abnormal number and morphology of cell nuclei were noticed in a subset of Cre-expressing RPE cells in aged heterozygous VMD2-Cre mice, whereas more severe nuclear abnormalities were observed already in young homozygous VMD2-Cre mice. Thus, continuous postnatal expression of Cre causes abnormalities in the RPE in an age- and Cre gene dosage-dependent manner, which needs to be considered in the interpretation of gene targeting studies in the RPE.

Progressive dysfunction of the retinal pigment epithelium and retina due to increased VEGF-A levels.

Patients with nonexudative ("dry") age-related macular degeneration (AMD) frequently also develop neovascular ("wet") AMD, suggesting a common pathomechanism. Increased vascular endothelial growth factor A (VEGF-A) has been implicated in the pathogenesis of choroidal neovascularization (CNV) in neovascular AMD, while its role in nonexudative AMD that manifests with progressive retinal pigment epithelium (RPE) and photoreceptor degeneration is not well defined. Mice with overall increased VEGF-A levels develop progressive morphological features of both forms of AMD, suggesting that an increase in VEGF-A has a direct age-dependent adverse effect on RPE and photoreceptor function independently of its CNV-promoting proangiogenic effect. Here we provide evidence for this hypothesis and show that morphological RPE abnormalities and retinal thinning in mice with increased VEGF-A levels correlate with progressive age-dependent attenuation of visual function with abnormal electroretinograms and reduced retinal rhodopsin levels. Retinoid profiling revealed a progressive reduction of 11-cis and all-trans retinal in the retinas of these mice, consistent with an impaired retinoid transport between the RPE and photoreceptors. These findings suggest that increased VEGF-A leads to an age-dependent RPE and retinal dysfunction that occurs also at sites where no CNV lesions form. The data support a central role of increased VEGF-A not only in the pathogenesis of neovascular but also of nonexudative AMD.

Macrophages are essential for the early wound healing response and the formation of a fibrovascular scar.

After wounding, multiple cell types interact to form a fibrovascular scar; the formation and cellular origins of these scars are incompletely understood. We used a laser-injury wound model of choroidal neovascularization in the eye to determine the spatiotemporal cellular events that lead to formation of a fibrovascular scar. After laser injury, F4/80(+) myeloid cells infiltrate the wound site and induce smooth muscle actin (SMA) expression in adjacent retinal pigment epithelial cells, with subsequent formation of a SMA(+)NG2(+) myofibroblastic scaffold, into which endothelial cells then infiltrate to form a fibrovascular lesion. Cells of the fibrovascular scaffold express the proangiogenic factor IL-1ß strongly, whereas retinal pigment epithelial cells are the main source of VEGF-A. Subsequent choroidal neovascularization is limited to the area demarcated by this myofibroblastic scaffold and occurs independently of epithelial- or myeloid-derived VEGF-A. The SMA(+)NG2(+) myofibroblastic cells, F4/80(+) macrophages, and adjacent epithelial cells actively proliferate in the early phase of the wound healing response. Cell-lineage tracing experiments suggest that the SMA(+)NG2(+) myofibroblastic scaffold originates from choroidal pericyte-like cells. Targeted ablation of macrophages inhibits the formation of this fibrovascular scaffold, and expression analysis reveals that these macrophages are Arg1(+)YM1(+)F4/80(+) alternatively activated M2-like macrophages, which do not require IL-4/STAT6 or IL-10 signaling for their activation. Thus, macrophages are essential for the early wound healing response and the formation of a fibrovascular scar.

Aplasia Cutis Congenita Pathomechanisms Reveal Key Regulators of Skin and Skin Appendage Morphogenesis.

Aplasia cutis congenita (ACC) manifests at birth as a defect of the scalp skin. New findings answer 2 longstanding questions: why ACC forms and why it affects mainly the midline scalp skin. Dominant-negative mutations in the genes KCTD1 or KCTD15 cause ACC owing to loss of function of KCTD1/KCTD15 complexes in cranial neural crest cells (NCCs), which normally form midline cranial suture mesenchymal cells that express keratinocyte growth factors. Loss of KCTD1/KCTD15 function in NCCs impairs the formation of normal midline cranial sutures and, consequently, the overlying skin, resulting in ACC. Moreover, KCTD1/KCTD15 complexes in keratinocytes regulate skin appendage morphogenesis.

AP-2α/AP-2ß Transcription Factors Are Key Regulators of Epidermal Homeostasis.

AP-2 transcription factors regulate ectodermal development, but their roles in epidermal homeostasis in adult skin are unknown. We find that AP-2α is the predominant AP-2 family member in adult epidermis, followed by AP-2ß. Through inactivation of AP-2α, AP-2ß, or both in keratinocytes, we assessed the effects of a gradient of epidermal AP-2 activity on skin function. We find that (i) loss of AP-2ß in keratinocytes is compensated for by AP-2α, (ii) loss of AP-2α impairs terminal keratinocyte differentiation and hair morphogenesis, and (iii) the combined loss of AP-2α/AP-2ß results in more severe skin and hair abnormalities. Keratinocyte differentiation defects precede progressive neutrophilic skin inflammation. Inducible inactivation of AP-2α/AP-2ß in the adult phenocopies these manifestations. Transcriptomic analyses of epidermis lacking AP-2α or AP-2α/AP-2ß in keratinocytes demonstrate a terminal keratinocyte differentiation defect with upregulation of alarmin keratins and of several immune pathway regulators. Moreover, our analyses suggest a key role of reduced AP-2α-dependent gene expression of CXCL14 and the keratin 15 gene K15 as an early pathogenic event toward the manifestation of skin inflammation. Thus, AP-2α and AP-2ß are critical regulators of epidermal homeostasis in adult skin.

Role of inflammasome activation in neovascular age-related macular degeneration.

Current anti-VEGF-A therapies inhibit choroidal neovascularization (CNV) in a subset of patients with neovascular age-related macular degeneration (NV-AMD). However, long-term treatment with such anti-VEGF-A therapies may impair physiological functions of the choriocapillaris and retina for which VEGF-A is needed. Moreover, disease progression can occur despite continuous anti-VEGF-A treatment. Thus, novel therapies for NV-AMD are urgently needed that target specifically disease-associated mechanisms without impairing growth factors and cellular pathways that are required for homeostatic functions of the retina and choroid. Inhibiting the inflammatory pathways that promote CNV would be such a promising novel approach that would likely not interfere with the normal functions of healthy retinal and choroidal cells. In this context, the inflammasome, a proinflammatory protein complex that promotes pathologic angiogenesis largely through generation of IL-1ß and which has been reported to be activated in AMD, has become an area of much interest in the AMD field. However, most studies have focused mainly on the NLRP3 inflammasome in retinal pigment epithelial cells (RPE), and conflicting findings have resulted in an unclear picture of the role of the inflammasome for AMD pathogenesis. Recent data suggest that inflammasome activation in activated macrophages and retinal microglia but not in RPE cells promotes CNV. Furthermore, inflammasome activation can occur in CNV macrophages and microglia despite lack of NLRP3. Thus, activation of both NLRP3 inflammasomes as well as non-NLRP3 inflammasomes in macrophages/microglia at sites of CNV formation likely promote NV-AMD.

AP-2α/AP-2ß transcription factors are key regulators of epidermal homeostasis.

AP-2 transcription factors regulate ectodermal development but their roles for epidermal homeostasis in the adult skin are unknown. We find that AP-2α is the predominant AP-2 family member in adult epidermis, followed by AP-2ß. Through inactivation of AP-2α, AP-2ß, or both in keratinocytes we assessed the effects of a gradient of epidermal AP-2 activity on skin function. We find that (1) loss of AP-2ß in keratinocytes is compensated for by AP-2α, (2) loss of AP-2α impairs terminal keratinocyte differentiation and hair morphogenesis, and (3) the combined loss of AP-2α/AP-2ß results in more severe skin and hair abnormalities. Keratinocyte differentiation defects precede a progressive neutrophilic skin inflammation. Inducible inactivation of AP-2α/AP-2ß in the adult phenocopies these manifestations. Transcriptomic analyses of epidermis lacking AP-2α or AP-2α/AP-2ß in keratinocytes demonstrate a terminal keratinocyte differentiation defect with upregulation of alarmin keratins and of several immune pathway regulators. Moreover, our analyses suggest a key role of loss of AP-2α-dependent gene expression of CXCL14 and KRT15 as an early pathogenic event towards the manifestation of skin inflammation. Thus, AP-2α/AP-2ß are critical regulators of epidermal homeostasis in the adult skin.

KCTD1/KCTD15 complexes control ectodermal and neural crest cell functions, and their impairment causes aplasia cutis.

Aplasia cutis congenita (ACC) is a congenital epidermal defect of the midline scalp and has been proposed to be due to a primary keratinocyte abnormality. Why it forms mainly at this anatomic site has remained a long-standing enigma. KCTD1 mutations cause ACC, ectodermal abnormalities, and kidney fibrosis, whereas KCTD15 mutations cause ACC and cardiac outflow tract abnormalities. Here, we found that KCTD1 and KCTD15 can form multimeric complexes and can compensate for each other's loss and that disease mutations are dominant negative, resulting in lack of KCTD1/KCTD15 function. We demonstrated that KCTD15 is critical for cardiac outflow tract development, whereas KCTD1 regulates distal nephron function. Combined inactivation of KCTD1/KCTD15 in keratinocytes resulted in abnormal skin appendages but not in ACC. Instead, KCTD1/KCTD15 inactivation in neural crest cells resulted in ACC linked to midline skull defects, demonstrating that ACC is not caused by a primary defect in keratinocytes but is a secondary consequence of impaired cranial neural crest cells, giving rise to midline cranial suture cells that express keratinocyte-promoting growth factors. Our findings explain the clinical observations in patients with KCTD1 versus KCTD15 mutations, establish KCTD1/KCTD15 complexes as critical regulators of ectodermal and neural crest cell functions, and define ACC as a neurocristopathy.

Protein kinase A activation inhibits oncogenic Sonic hedgehog signalling and suppresses basal cell carcinoma of the skin.

Basal cell carcinoma of the skin (BCC) is caused by constitutive activation of the Sonic hedgehog (Shh) pathway, mainly through mutations either in the Shh receptor Patched (PTCH) or in its co-receptor Smoothened (Smo). Inhibitors of this pathway that are currently in clinical trials inhibit Smo. However, mutations in Smo can result in resistance to these inhibitors. To target most BCCs and avoid acquired resistance because of Smo mutations, inhibiting the Shh-pathway downstream of Smo is critical. Attractive downstream targets would be at the level of Gli proteins, the transcriptional activators of this pathway in BCCs. Previously it has been shown that Gli1 and Gli2, when phosphorylated by protein kinase A (PKA), are targeted for proteosomal degradation. Here we show that PKA activation via the cAMP agonist forskolin is sufficient to completely abolish oncogenic Smo activity in vitro. In an inducible BCC mouse model due to a Smo mutation that confers resistance to current Smo inhibitors, topical forskolin treatment significantly reduced Gli1 mRNA levels and resulted in strongly suppressed BCC tumor growth. Our data show that forskolin inhibits the growth of even those BCCs that are resistant to Smo inhibitors and provide a proof-of-principle framework for the development of topically applied human skin-permeable novel pharmacologic inhibitors of oncogenic Shh-signaling through PKA activation.

Transcription factors AP-2α and AP-2ß regulate distinct segments of the distal nephron in the mammalian kidney.

Transcription factors AP-2α and AP-2ß have been suggested to regulate the differentiation of nephron precursor populations towards distal nephron segments. Here, we show that in the adult mammalian kidney AP-2α is found in medullary collecting ducts, whereas AP-2ß is found in distal nephron segments except for medullary collecting ducts. Inactivation of AP-2α in nephron progenitor cells does not affect mammalian nephrogenesis, whereas its inactivation in collecting ducts leads to defects in medullary collecting ducts in the adult. Heterozygosity for AP-2ß in nephron progenitor cells leads to progressive distal convoluted tubule abnormalities and ß-catenin/mTOR hyperactivation that is associated with renal fibrosis and cysts. Complete loss of AP-2ß in nephron progenitor cells caused an absence of distal convoluted tubules, renal cysts, and fibrosis with ß-catenin/mTOR hyperactivation, and early postnatal death. Thus, AP-2α and AP-2ß have non-redundant distinct spatiotemporal functions in separate segments of the distal nephron in the mammalian kidney.

Pralatrexate-induced tumor cell apoptosis in the epidermis of a patient with HTLV-1 adult T-cell lymphoma/leukemia causing skin erosions.

Pralatrexate is a novel antifolate, which shows increased antitumor activity in human tumor xenograft studies in mice compared with methotrexate. We investigated the effects of pralatrexate in a patient with adult T-cell lymphoma/leukemia with significant skin involvement. Atypical lymphocytes in epidermal Pautrier microabscesses were positive for HTLV-1. After the patient presented with leukemic conversion and with worsening of an erythematous generalized papular rash, he received one dose of pralatrexate. Within one week, his skin developed innumerable small erosions limited to the areas of the papular rash, sparing unaffected skin. Here we present in vivo evidence that pralatrexate-induced erosions in skin affected by adult T-cell lymphoma/leukemia are a manifestation of apoptosis of tumor cells infiltrating the epidermis and are not the result of cytotoxicity by pralatrexate on keratinocytes. This distinction is critical and may profoundly influence the clinical decision to continue pralatrexate treatment. Pralatrexate-induced skin erosions may indicate response to treatment.