Investigation into the genetics of fetal congenital lymphatic anomalies.

OBJECTIVE: Congenital lymphatic anomalies (LAs) arise due to defects in lymphatic development and often present in utero as pleural effusion, chylothorax, nuchal and soft tissue edema, ascites, or hydrops. Many LAs are caused by single nucleotide variants, which are not detected on routine prenatal testing. METHODS: Demographic data were compared between two subcohorts, those with clinically significant fetal edema (CSFE) and isolated fetal edema. A targeted variant analysis of LA genes was performed using American College of Medical Genetics criteria on whole exome sequencing (WES) data generated for 71 fetal edema cases who remained undiagnosed after standard workup. RESULTS: CSFE cases had poor outcomes, including preterm delivery, demise, and maternal preeclampsia. Pathogenic and likely pathogenic variants were identified in 7% (5/71) of cases, including variants in RASopathy genes, RASA1, SOS1, PTPN11, and a novel PIEZO1 variant. Variants of uncertain significance (VOUS) were identified in 45% (32/71) of cases. In CSFEs, VOUS were found in CELSR1, EPHB4, TIE1, PIEZO1, ITGA9, RASopathy genes, SOS1, SOS2, and RAF1. CONCLUSIONS: WES identified pathogenic and likely pathogenic variants and VOUS in LA genes in 51% of fetal edema cases, supporting WES and expanded hydrops panels in cases of idiopathic fetal hydrops and fluid collections.

Unique functions for Notch4 in murine embryonic lymphangiogenesis.

In mice, embryonic dermal lymphatic development is well understood and used to study gene functions in lymphangiogenesis. Notch signaling is an evolutionarily conserved pathway that modulates cell fate decisions, which has been shown to both inhibit and promote dermal lymphangiogenesis. Here, we demonstrate distinct roles for Notch4 signaling versus canonical Notch signaling in embryonic dermal lymphangiogenesis. Actively growing embryonic dermal lymphatics expressed NOTCH1, NOTCH4, and DLL4 which correlated with Notch activity. In lymphatic endothelial cells (LECs), DLL4 activation of Notch induced a subset of Notch effectors and lymphatic genes, which were distinctly regulated by Notch1 and Notch4 activation. Treatment of LECs with VEGF-A or VEGF-C upregulated Dll4 transcripts and differentially and temporally regulated the expression of Notch1 and Hes/Hey genes. Mice nullizygous for Notch4 had an increase in the closure of the lymphangiogenic fronts which correlated with reduced vessel caliber in the maturing lymphatic plexus at E14.5 and reduced branching at E16.5. Activation of Notch4 suppressed LEC migration in a wounding assay significantly more than Notch1, suggesting a dominant role for Notch4 in regulating LEC migration. Unlike Notch4 nulls, inhibition of canonical Notch signaling by expressing a dominant negative form of MAML1 (DNMAML) in Prox1+ LECs led to increased lymphatic density consistent with an increase in LEC proliferation, described for the loss of LEC Notch1. Moreover, loss of Notch4 did not affect LEC canonical Notch signaling. Thus, we propose that Notch4 signaling and canonical Notch signaling have distinct functions in the coordination of embryonic dermal lymphangiogenesis.

Notch Signaling Mediates Differentiation in Barrett's Esophagus and Promotes Progression to Adenocarcinoma.

BACKGROUND & AIMS: Studies are needed to determine the mechanism by which Barrett's esophagus (BE) progresses to esophageal adenocarcinoma (EAC). Notch signaling maintains stem cells in the gastrointestinal tract and is dysregulated during carcinogenesis. We explored the relationship between Notch signaling and goblet cell maturation, a feature of BE, during EAC pathogenesis. METHODS: We measured goblet cell density and levels of Notch messenger RNAs in BE tissues from 164 patients, with and without dysplasia or EAC, enrolled in a multicenter study. We analyzed the effects of conditional expression of an activated form of NOTCH2 (pL2.Lgr5.N2IC), conditional deletion of NOTCH2 (pL2.Lgr5.N2fl/fl), or loss of nuclear factor κB (NF-κB) (pL2.Lgr5.p65fl/fl), in Lgr5+ (progenitor) cells in L2-IL1B mice (which overexpress interleukin 1 beta in esophagus and squamous forestomach and are used as a model of BE). We collected esophageal and stomach tissues and performed histology, immunohistochemistry, flow cytometry, transcriptome, and real-time polymerase chain reaction analyses. Cardia and forestomach tissues from mice were cultured as organoids and incubated with inhibitors of Notch or NF-kB. RESULTS: Progression of BE to EAC was associated with a significant reduction in goblet cell density comparing nondysplastic regions of tissues from patients; there was an inverse correlation between goblet cell density and levels of NOTCH3 and JAG2 messenger RNA. In mice, expression of the activated intracellular form of NOTCH2 in Lgr5+ cells reduced goblet-like cell maturation, increased crypt fission, and accelerated the development of tumors in the squamocolumnar junction. Mice with deletion of NOTCH2 from Lgr5+ cells had increased maturation of goblet-like cells, reduced crypt fission, and developed fewer tumors. Esophageal tissues from in pL2.Lgr5.N2IC mice had increased levels of RelA (which encodes the p65 unit of NF-κB) compared to tissues from L2-IL1B mice, and we found evidence of increased NF-κB activity in Lgr5+ cells. Esophageal tissues from pL2.Lgr5.p65fl/fl mice had lower inflammation and metaplasia scores than pL2.Lgr5.N2IC mice. In organoids derived from pL2-IL1B mice, the NF-κB inhibitor JSH-23 reduced cell survival and proliferation. CONCLUSIONS: Notch signaling contributes to activation of NF-κB and regulates differentiation of gastric cardia progenitor cells in a mouse model of BE. In human esophageal tissues, progression of BE to EAC was associated with reduced goblet cell density and increased levels of Notch expression. Strategies to block this pathway might be developed to prevent EAC in patients with BE.

Implications for preeclampsia: hypoxia-induced Notch promotes trophoblast migration.

In the first trimester of human pregnancy, low oxygen tension or hypoxia is essential for proper placentation and placenta function. Low oxygen levels and activation of signaling pathways have been implicated as critical mediators in the promotion of trophoblast differentiation, migration, and invasion with inappropriate changes in oxygen tension and aberrant Notch signaling both individually reported as causative to abnormal placentation. Despite crosstalk between hypoxia and Notch signaling in multiple cell types, the relationship between hypoxia and Notch in first trimester trophoblast function is not understood. To determine how a low oxygen environment impacts Notch signaling and cellular motility, we utilized the human first trimester trophoblast cell line, HTR-8/SVneo. Gene set enrichment and ontology analyses identified pathways involved in angiogenesis, Notch and cellular migration as upregulated in HTR-8/SVneo cells exposed to hypoxic conditions. DAPT, a γ-secretase inhibitor that inhibits Notch activation, was used to interrogate the crosstalk between Notch and hypoxia pathways in HTR-8/SVneo cells. We found that hypoxia requires Notch activation to mediate HTR-8/SVneo cell migration, but not invasion. To determine if our in vitro findings were associated with preeclampsia, we analyzed the second trimester chorionic villous sampling (CVS) samples and third trimester placentas. We found a significant decrease in expression of migration and invasion genes in CVS from preeclamptic pregnancies and significantly lower levels of JAG1 in placentas from pregnancies with early-onset preeclampsia with severe features. Our data support a role for Notch in mediating hypoxia-induced trophoblast migration, which may contribute to preeclampsia development.

Evidence of vasculature and chondrocyte to osteoblast transdifferentiation in craniofacial synovial joints: Implications for osteoarthritis diagnosis and therapy.

Temporomandibular joint osteoarthritis (TMJ OA) leads to permanent cartilage destruction, jaw dysfunction, and compromises the quality of life. However, the pathological mechanisms governing TMJ OA are poorly understood. Unlike appendicular articular cartilage, the TMJ has two distinct functions as the synovial joint of the craniofacial complex and also as the site for endochondral jaw bone growth. The established dogma of endochondral bone ossification is that hypertrophic chondrocytes undergo apoptosis, while invading vasculature with osteoprogenitors replace cartilage with bone. However, contemporary murine genetic studies support the direct differentiation of chondrocytes into osteoblasts and osteocytes in the TMJ. Here we sought to characterize putative vasculature and cartilage to bone transdifferentiation using healthy and diseased TMJ tissues from miniature pigs and humans. During endochondral ossification, the presence of fully formed vasculature expressing CD31+ endothelial cells and α-SMA+ vascular smooth muscle cells were detected within all cellular zones in growing miniature pigs. Arterial, endothelial, venous, angiogenic, and mural cell markers were significantly upregulated in miniature pig TMJ tissues relative to donor matched knee meniscus fibrocartilage tissue. Upon surgically creating TMJ OA in miniature pigs, we discovered increased vasculature and putative chondrocyte to osteoblast transformation dually marked by COL2 and BSP or RUNX2 within the vascular bundles. Pathological human TMJ tissues also exhibited increased vasculature, while isolated diseased human TMJ cells exhibited marked increased in vasculature markers relative to control 293T cells. Our study provides evidence to suggest that the TMJ in higher order species are in fact vascularized. There have been no reports of cartilage to bone transdifferentiation or vasculature in human-relevant TMJ OA large animal models or in human TMJ tissues and cells. Therefore, these findings may potentially alter the clinical management of TMJ OA by defining new drugs that target angiogenesis or block the cartilage to bone transformation.

Endothelial Jagged1 Antagonizes Dll4/Notch Signaling in Decidual Angiogenesis during Early Mouse Pregnancy.

Maternal spiral arteries and newly formed decidual capillaries support embryonic development prior to placentation. Previous studies demonstrated that Notch signaling is active in endothelial cells of both decidual capillaries and spiral arteries, however the role of Notch signaling in physiologic decidual angiogenesis and maintenance of the decidual vasculature in early mouse pregnancy has not yet been fully elucidated. We used the Cdh5-CreERT2;Jagged1(Jag1)flox/flox (Jag1∆EC) mouse model to delete Notch ligand, Jag1, in maternal endothelial cells during post-implantation, pre-placentation mouse pregnancy. Loss of endothelial Jag1 leads to increased expression of Notch effectors, Hey2 and Nrarp, and increased endothelial Notch signaling activity in areas of the decidua with remodeling angiogenesis. This correlated with an increase in Dll4 expression in capillary endothelial cells, but not spiral artery endothelial cells. Consistent with increased Dll4/Notch signaling, we observed decreased VEGFR2 expression and endothelial cell proliferation in angiogenic decidual capillaries. Despite aberrant Dll4 expression and Notch activation in Jag1∆EC mutants, pregnancies were maintained and the decidual vasculature was not altered up to embryonic day 7.5. Thus, Jag1 functions in the newly formed decidual capillaries as an antagonist of endothelial Dll4/Notch signaling during angiogenesis, but Jag1 signaling is not necessary for early uterine angiogenesis.

Angiogenic factor imbalance precedes complement deposition in placentae of the BPH/5 model of preeclampsia.

Preeclampsia (PE), a hypertensive disorder of pregnancy, is a leading cause of maternal and fetal morbidity and mortality. Although the etiology is unknown, PE is thought to be caused by defective implantation and decidualization in pregnancy. Pregnant blood pressure high (BPH)/5 mice spontaneously develop placentopathies and maternal features of human PE. We hypothesized that BPH/5 implantation sites have transcriptomic alterations. Next-generation RNA sequencing of implantation sites at peak decidualization, embryonic day (E)7.5, revealed complement gene up-regulation in BPH/5 vs. controls. In BPH/5, expression of complement factor 3 was increased around the decidual vasculature of E7.5 implantation sites and in the trophoblast giant cell layer of E10.5 placentae. Altered expression of VEGF pathway genes in E5.5 BPH/5 implantation sites preceded complement dysregulation, which correlated with abnormal vasculature and increased placental growth factor mRNA and VEGF164 expression at E7.5. By E10.5, proangiogenic genes were down-regulated, whereas antiangiogenic sFlt-1 was up-regulated in BPH/5 placentae. We found that early local misexpression of VEGF genes and abnormal decidual vasculature preceded sFlt-1 overexpression and increased complement deposition in BPH/5 placentae. Our findings suggest that abnormal decidual angiogenesis precedes complement activation, which in turn contributes to the aberrant trophoblast invasion and poor placentation that underlie PE.-Sones, J. L., Merriam, A. A., Seffens, A., Brown-Grant, D.-A., Butler, S. D., Zhao, A. M., Xu, X., Shawber, C. J., Grenier, J. K., Douglas, N. C. Angiogenic factor imbalance precedes complement deposition in placentae of the BPH/5 model of preeclampsia.

Notch signaling functions in lymphatic valve formation.

Collecting lymphatic ducts contain intraluminal valves that prevent backflow. In mice, lymphatic valve morphogenesis begins at embryonic day 15.5 (E15.5). In the mesentery, Prox1 expression is high in valve-forming lymphatic endothelial cells, whereas cells of the lymphatic ducts express lower levels of Prox1. Integrin α9, fibronectin EIIIA, Foxc2, calcineurin and the gap junction protein Cx37 are required for lymphatic valve formation. We show that Notch1 is expressed throughout the developing mesenteric lymphatic vessels at E16.5, and that, by E18.5, Notch1 expression becomes highly enriched in the lymphatic valve endothelial cells. Using a Notch reporter mouse, Notch activity was detected in lymphatic valves at E17.5 and E18.5. The role of Notch in lymphatic valve morphogenesis was studied using a conditional lymphatic endothelial cell driver either to delete Notch1 or to express a dominant-negative Mastermind-like (DNMAML) transgene. Deletion of Notch1 led to an expansion of Prox1(high) cells, a defect in Prox1(high) cell reorientation and a decrease in integrin α9 expression at sites of valve formation. Expression of DNMAML, which blocks all Notch signaling, resulted in a more severe phenotype characterized by a decrease in valves, failure of Prox1(high) cells to cluster, and rounding of the nuclei and decreased fibronectin-EIIIA expression in the Prox1(high) cells found at valve sites. In human dermal lymphatic endothelial cells, activation of Notch1 or Notch4 induced integrin α9, fibronectin EIIIA and Cx37 expression. We conclude that Notch signaling is required for proper lymphatic valve formation and regulates integrin α9 and fibronectin EIIIA expression during valve morphogenesis.

Notch1 functions as a negative regulator of lymphatic endothelial cell differentiation in the venous endothelium.

In development, lymphatic endothelial cells originate within veins and differentiate via a process requiring Prox1. Notch signaling regulates cell-fate decisions, and expression studies suggested that Jag1/Notch1 signaling functions in veins during lymphatic endothelial specification. Using an inducible lymphatic endothelial Prox1CreER(T2) driver, Notch signaling was suppressed by deleting Notch1 or expressing dominant-negative Mastermind-like in Prox1+ endothelial cells. Either loss of Notch1 or reduced Notch signaling increased Prox1+ lymphatic endothelial progenitor cell numbers in the veins, leading to incomplete separation of venous and lymphatic vessels. Notch loss of function resulted in excessive Prox1+ lymphatic cells emerging from the cardinal vein and significant lymphatic overgrowth. Moreover, loss of one allele of Notch1 in Prox1 heterozygous mice rescued embryonic lethality due to Prox1 haploinsufficiency and significantly increased Prox1+ lymphatic endothelial progenitor cell numbers. Expression of a constitutively active Notch1 protein in Prox1+ cells suppressed endothelial Prox1 from E9.75 to E13.5, resulting in misspecified lymphatic endothelial cells based upon reduced expression of podoplanin, LYVE1 and VEGFR3. Notch activation resulted in the appearance of blood endothelial cells in peripheral lymphatic vessels. Activation of Notch signaling in the venous endothelium at E10.5 did not arterialize the cardinal vein, suggesting that Notch can no longer promote arterialization in the cardinal vein during this developmental stage. We report a novel role for Notch1 in limiting the number of lymphatic endothelial cells that differentiate from the veins to assure proper lymphatic specification.

A new paradigm for the role of smooth muscle cells in the human cervix.

BACKGROUND: Premature cervical remodeling resulting in spontaneous preterm birth may begin with premature failure or relaxation at the internal os (termed "funneling"). To date, we do not understand why the internal os fails or why funneling occurs in some cases of premature cervical remodeling. Although the human cervix is thought to be mostly collagen with minimal cellular content, cervical smooth muscle cells are present in the cervix and can cause cervical tissue contractility. OBJECTIVE: To understand why the internal os relaxes or why funneling occurs in some cases of premature cervical remodeling, we sought to evaluate cervical smooth muscle cell content and distribution throughout human cervix and correlate if cervical smooth muscle organization influences regional cervical tissue contractility. STUDY DESIGN: Using institutional review board-approved protocols, nonpregnant women <50 years old undergoing hysterectomy for benign indications were consented. Cervical tissue from the internal and external os were immunostained for smooth muscle cell markers (α-smooth muscle actin, smooth muscle protein 22 calponin) and contraction-associated proteins (connexin 43, cyclooxygenase-2, oxytocin receptor). To evaluate cervical smooth muscle cell morphology throughout the entire cervix, whole cervical slices were obtained from the internal os, midcervix, and external os and immunostained with smooth muscle actin. To correlate tissue structure with function, whole slices from the internal and external os were stimulated to contract with 1 µmol/L of oxytocin in organ baths. In separate samples, we tested if the cervix responds to a common tocolytic, nifedipine. Cervical slices from the internal os were treated with oxytocin alone or oxytocin + increasing doses of nifedipine to generate a dose response and half maximal inhibitory concentration. Student t test was used where appropriate. RESULTS: Cervical tissue was collected from 41 women. Immunohistochemistry showed cervical smooth muscle cells at the internal and external os expressed mature smooth muscle cell markers and contraction-associated proteins. The cervix exhibited a gradient of cervical smooth muscle cells. The area of the internal os contained 50-60% cervical smooth muscle cells that were circumferentially organized in the periphery of the stroma, which may resemble a sphincter-like pattern. The external os contained approximately 10% cervical smooth muscle cells that were randomly scattered in the tissue. In organ bath studies, oxytocin stimulated the internal os to contract with more than double the force of the external os (1341 ± 693 vs 523 ± 536 integrated grams × seconds, respectively, P = .009). Nifedipine significantly decreased cervical tissue muscle force compared to timed vehicle control (oxytocin alone) at doses of 10(-5) mol/L (vehicle 47% ± 15% vs oxytocin + nifedipine 24% ± 16%, P = .007), 10(-4) mol/L (vehicle 46% ± 16% vs oxytocin + nifedipine -4% ± 20%, P = .003), and 10(-3) mol/L (vehicle 42% ± 14% vs oxytocin + nifedipine -15% ± 18%, P = .0006). The half maximal inhibitory concentration for nifedipine was 1.35 × 10(-5) mol/L. CONCLUSION: Our findings suggest a new paradigm for cervical tissue morphology-one that includes the possibility of a specialized sphincter at the internal os. This new paradigm introduces novel avenues to further investigate potential mechanisms of normal and premature cervical remodeling.

Growth Hormone Induces Recurrence of Infantile Hemangiomas After Apparent Involution: Evidence of Growth Hormone Receptors in Infantile Hemangioma.

Infantile hemangiomas (IHs) are the most common benign tumor of infancy, characterized by a natural history of early proliferation in the first months of life to eventual involution during childhood, often with residual fibrofatty tissue. Once involution has been achieved, IHs do not typically recur. We present two cases of exogenous growth hormone therapy resulting in the recurrence of IHs in late childhood, supported by radiological, immunohistochemical, in vitro, and in vivo evidence.

Propranolol promotes accelerated and dysregulated adipogenesis in hemangioma stem cells.

BACKGROUND: Infantile hemangiomas (IHs) are the most common tumor of infancy, yet there are no Food and Drug Administration-approved therapeutics to date. Recently, the nonselective ß-adrenergic-blocker propranolol has been shown to be a safe and effective means of treating IHs, although its mechanism has yet to be elucidated. We have previously demonstrated that propranolol induces early and incomplete adipogenesis in stem cells derived from hemangiomas. We hypothesize that propranolol promotes dysregulated adipogenesis via the improper regulation of adipogenic genes. METHODS: Hemangioma stem cells (HemSCs) isolated from resected IH specimens were treated with adipogenic medium for 1 or 4 days in either propranolol or vehicle. Cell death was measured by the incorporation of annexin V and propidium iodide by flow cytometry. Adipogenesis was assessed by visualizing lipid droplet formation by Oil Red O staining. Proadipogenic genes C/EBPα, C/EBPß, C/EBPδ, PPARδ, PPARγ, RXRα, and RXRγ were analyzed by quantitative reverse transcription and polymerase chain reaction. RESULTS: Hemangioma stem cells treated with propranolol increased lipid droplet formation compared to vehicle-treated cells indicating increased adipogenesis. Cell death as measured by FACS analysis indicated that the propranolol-treated cells died due to necrosis and not apoptosis. During adipogenesis, transcript levels of PPARδ, PPARγ, C/EBPß, and C/EBPδ were significantly increased (P<0.01) in propranolol-treated cells relative to control cells. In contrast, RXRα and RXRγ levels were significantly decreased (P<0.05), and C/EBPα, a gene required for terminal adipocyte differentiation, was strongly suppressed by propranolol when compared to vehicle-treated cells (P<0.01). CONCLUSIONS: In HemSCs, propranolol accelerated dysregulated adipogenic differentiation characterized by improper adipogenic gene expression. Consistent with accelerated adipogenesis, propranolol significantly increased the expression of the proadipogenic genes, PPARγ, C/EBPß, and C/EBPγ compared to control. However, propranolol treatment also led to improper induction of PPARδ and suppression of C/EBPα, RXRα, and RXRγ. Taken together these data indicate that propranolol promoted dysregulated adipogenesis and inhibited the HemSCs from becoming functional adipocytes, ultimately resulting in cell death. Understanding this mechanism behind propranolol's effectiveness will be a vital factor in producing more effective therapies in the future.

Microbial-Dependent Recruitment of Immature Myeloid Cells Promotes Intestinal Regeneration.

BACKGROUND & AIMS: The intestinal epithelium functions both in nutrient absorption and as a barrier, separating the luminal contents from a network of vascular, fibroblastic, and immune cells underneath. After injury to the intestine, multiple cell populations cooperate to drive regeneration of the mucosal barrier, including lymphatic endothelial cells (LECs). A population of granulocytic immature myeloid cells (IMCs), marked by Hdc, participate in regeneration of multiple organs such as the colon and central nervous system, and their contribution to intestinal regeneration was investigated. METHODS: By using male and female histidine decarboxylase (Hdc) green fluorescent reporter (GFP) mice, we investigated the role of Hdc+ IMCs in intestinal regeneration after exposure to 12 Gy whole-body irradiation. The movement of IMCs was analyzed using flow cytometry and immunostaining. Ablation of Hdc+ cells using the HdcCreERT2 tamoxifen-inducible recombinase Cre system, conditional knockout of Prostaglandin-endoperoxidase synthase 2 (Ptgs2) in Hdc+ cells using HdcCre; Ptgs2 floxed mice, and visualization of LECs using Prox1tdTomato mice also was performed. The role of microbial signals was investigated by knocking down mice gut microbiomes using antibiotic cocktail gavages. RESULTS: We found that Hdc+ IMCs infiltrate the injured intestine after irradiation injury and promote epithelial regeneration in part by modulating LEC activity. Hdc+ IMCs express Ptgs2 (encoding cyclooxygenase-2/COX-2), and enables them to produce prostaglandin E2. Prostaglandin E2 acts on the prostaglandin E2 receptor 4 receptor (EP4) on LECs to promote lymphangiogenesis and induce the expression of proregenerative factors including R-spondin 3. Depletion of gut microbes leads to reduced intestinal regeneration by impaired recruitment of IMCs. CONCLUSIONS: Altogether, our results unveil a critical role for IMCs in intestinal repair by modulating LEC activity and implicate gut microbes as mediators of intestinal regeneration.

Intraovarian regulation of gonadotropin-dependent folliculogenesis depends on notch receptor signaling pathways not involving Delta-like ligand 4 (Dll4).

BACKGROUND: In-situ hybridisation studies demonstrate that Notch receptors and ligands are expressed in granulosa cells (GCs) and in the theca layer vasculature of growing follicles. Notch signaling involves cell-to-cell interaction mediated by transmembrane receptors and ligands. This signaling pathway may represent a novel intraovarian regulator of gonadotropin-dependent follicular development to the preovulatory stage. We hypothesized that blocking Notch pathways would disrupt follicular maturation in the mouse ovary. METHODS: Hypophysectomized CD21 female mice were administered pregnant mare serum gonadotropin (PMSG) for 3 days to stimulate follicular development. In one experiment, a pan-notch inhibitor, compound E, was initiated 2 days prior to and throughout stimulation (n = 10), while in a second experiment, a humanized phage Dll4 blocking antibody, YW152F, was used (n = 5). After sacrifice, ovarian histology, serum estradiol levels and uterine weights were compared to controls. The ovarian morphology was evaluated with hematoxylin/eosin staining and immunohistochemistry was performed for Notch1, Notch2, Notch3, Notch4, Jagged1, Dll4, platelet endothelial cell adhesion molecule (PECAM) and alpha-smooth muscle actin (α-SMA) detection. RESULTS: We localized specific Notch ligands and receptors in the following structures: Dll4 is specific to theca layer endothelial cells (ECs); Notch1/Notch4 and Jagged1 are expressed in theca layer ECs and vascular smooth muscle cells (VSMCs), whereas Notch3 is restricted to VSMCs; Notch2 is expressed mostly on GCs of small follicles. Administration of a pan-Notch inhibitor, compound E, inhibits follicular development to the preovulatory stage (8.5 preovulatory follicles in treatment vs. 3.4 preovulatory follicles in control, p < 0.01; average number per ovary) with significant secondary effects on ovarian and uterine weight and estradiol secretion in a setting of uninhibited vascular proliferation, but disorganized appearance of ECs and VSMCs. Inhibition of endothelial Notch1 function through the inactivation of its ligand Dll4 with the blocking antibody YW152F induces mild disorganisation of follicular vasculature, but has no significant effect on gonadotropin-dependent folliculogenesis. CONCLUSIONS: Our experiments suggest that the complete blockage of the Notch signaling pathway with compound E impairs folliculogenesis and induces disruption of gonadotropin stimulated angiogenesis. It seems the mechanism involves Notch1 and Notch3, specifically, causing the improper assembly of ECs and VSMCs in the theca layer, although the potential role of non-angiogenic Notch signaling, such as Jagged2 to Notch2 in GCs, remains to be elucidated.

Pathophysiology of Slow-Flow Vascular Malformations: Current Understanding and Unanswered Questions.

Background: Slow-flow vascular malformations include venous, lymphatic, and lymphaticovenous malformations. Recent studies have linked genetic variants hyperactivating either the PI3K/AKT/mTOR and/or RAS/RAF/MAPK signaling pathways with slow-flow vascular malformation development, leading to the use of pharmacotherapies such as sirolimus and alpelisib. It is important that clinicians understand basic and translational research advances in slow-flow vascular malformations. Methods: A literature review of basic science publications in slow-flow vascular malformations was performed on Pubmed, using search terms "venous malformation," "lymphatic malformation," "lymphaticovenous malformation," "genetic variant," "genetic mutation," "endothelial cells," and "animal model." Relevant publications were reviewed and summarized. Results: The study of patient tissues and the use of primary pathogenic endothelial cells from vascular malformations shed light on their pathological behaviors, such as endothelial cell hyperproliferation and disruptions in vessel architecture. The use of xenograft and transgenic animal models confirmed the pathogenicity of genetic variants and allowed for preclinical testing of potential therapies. These discoveries underscore the importance of basic and translational research in understanding the pathophysiology of vascular malformations, which will allow for the development of improved biologically targeted treatments. Conclusion: Despite basic and translation advances, a cure for slow-flow vascular malformations remains elusive. Many questions remain unanswered, including how genotype variants result in phenotypes, and genotype-phenotype heterogeneity. Continued research into venous and lymphatic malformation pathobiology is critical in understanding the mechanisms by which genetic variants contribute to vascular malformation phenotypic features.

Propranolol Therapy for Congenital Chylothorax.

Congenital chylothorax is a rare and often severe anomaly without well-established medical therapies. Previously, propranolol use in patients with lymphatic malformations and secondary chylothorax was associated with improvement in clinical signs. We hypothesized that propranolol treatment would be beneficial for severe congenital chylothorax. We reviewed medical records of neonates born from 2015 to 2019 at our tertiary center with a prenatal diagnosis of congenital chylothorax for whom either prenatal or postnatal propranolol therapy was initiated. Inclusion was limited to fetuses diagnosed with severe congenital chylothorax without significant genetic, infectious, or cardiac anomalies, and who underwent prenatal interventions to mitigate consequences of the condition. Propranolol was administered orally to pregnant women at 20 mg 4 times daily and increased to a maximum dose of 40 mg 4 times daily, or to infants at 0.3 mg/kg/d and increased to 1 to 2 mg/kg/d. Primary outcomes were the time course of resolution of ultrasonographical, clinical, and/or radiologic signs of chylothorax after treatment with propranolol. Four neonates met the inclusion criteria. In 2 cases, prenatal initiation of propranolol led to resolution of the chylothoraxes before delivery (38 and 32 days after treatment) on a dose of 40 mg/day 4 times daily. Neonates had a normal postnatal course. Postnatal propranolol was initiated in 2 neonates with respiratory failure when chylothoraces were refractory to standard management. Stabilization and improvement of their pleural effusion was observed by imaging at 29 and 13 days after initiation of propranolol. There were no significant maternal or neonatal complications from prenatal or postnatal propranolol use. Propranolol may be efficacious in treating severe fetal congenital chylothorax.

Immature myeloid cells are indispensable for intestinal regeneration post irradiation injury.

The intestinal epithelium functions both in nutrient absorption and as a barrier, separating the luminal contents from a network of vascular, fibroblastic, and immune cells underneath. Following injury to the intestine, multiple different cell populations cooperate to drive regeneration of the mucosa. Immature myeloid cells (IMCs), marked by histidine decarboxylase ( Hdc ), participate in regeneration of multiple organs such as the colon and central nervous system. Here, we found that IMCs infiltrate the injured intestine and promote epithelial regeneration and modulate LEC activity. IMCs produce prostaglandin E2 (PGE2), which promotes LEC lymphangiogenesis and upregulation of pro-regenerative factors including RSPO3. Moreover, we found that IMC recruitment into the intestine is driven by invading microbial signals. Accordingly, antibiotic eradication of the intestinal microbiome prior to WB-IR inhibits IMC recruitment, and consequently, intestinal recovery. We propose that IMCs play a critical role in intestinal repair and implicate gut microbes as mediators of intestinal regeneration.

Propranolol treatment for chylothorax after congenital cardiac surgery.

OBJECTIVE: Postoperative chylothorax causes significant morbidities in pediatric patients with cardiac disease. New treatment approaches based on evolving understanding of underlying lymphatic dysfunction are being developed. We hypothesized that propranolol reduces morbidities and duration of chest tube requirement in high-output chylous effusion. METHODS: The postoperative courses of 50 pediatric patients with cardiac disease and high-output chylous effusion (control, n = 25; propranolol-treated, n = 25) were reviewed, including morbidities, length of hospitalization, and duration of chest tube requirement. Statistical analysis was performed using Welch's t test, Kruskal-Wallis tests for continuous variables, and chi-square and Fisher exact tests for categorical variables. Univariable logistic regression was used to determine predictors of response. RESULTS: Propranolol response was defined as 80% or more drainage reduction in 9 days or less. Treated patients were grouped into responders (<9 days) and nonresponders (>10 days). Neither initial amount of drainage (P = .12) nor day of propranolol initiation (P = .17) correlated with response. When compared with controls and nonresponders, responders had significantly fewer days with chest tube requirement (P < .01), infection (P < .0002), and thrombus (P = .005), and shorter hospitalization (P < .05). All patients had low serum albumin, although nonresponders had significantly decreased serum albumin when compared with responders and control patients (P < .002), and were more likely to receive albumin replacement (P < .01). Malnutrition was prevalent in all patient groups. CONCLUSIONS: Responders to propranolol had significantly less morbidity and duration of chest tube requirement when compared with control patients and nonresponders. Nonresponders did not have worse outcomes than control patients. We conclude that propranolol may be an effective treatment of patients with refractory chylothorax.

Inactivation of Notch4 Attenuated Pancreatic Tumorigenesis in Mice.

Expression of the Notch family of receptors is often upregulated in pancreatic ductal adenocarcinoma (PDAC). In this study, we focused on Notch4, which had not been investigated in PDAC. We generated KC (LSL-KrasG12D;p48-Cre), N4 - / - KC (Notch4- / -;LSL-KrasG12D;p48-Cre), PKC (p16fl/fl;LSL-KrasG12D;p48-Cre), and N4 - / - PKC (Notch4-/ -; p16fl/f l;LSL-KrasG12D;p48-Cre) genetically engineered mouse models (GEMM). We performed caerulein treatment in both KC and N4 - / - KC mice, and the development of acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) lesions were significantly diminished in the N4 - / - KC than in the KC GEMM (P = 0.01). This in vivo result was validated by in vitro ADM induction of the explant cultures of pancreatic acinar cells from the N4 - / - KC and KC mice (P < 0.001), confirming that Notch4 is an important contributor to early pancreatic tumorigenesis. To evaluate the role of Notch4 in the later stage of pancreatic tumorigenesis, we compared the PKC and N4 - / - PKC mice. The N4 - / - PKC mice had better overall survival (P = 0.012) and significantly reduced tumor burden (PanIN: P = 0.018 at 2 months, PDAC: P = 0.039 at 5 months) compared with the PKC GEMM. RNA-sequencing analysis of pancreatic tumor cell lines derived from the PKC and N4 - / - PKC GEMMs revealed that 408 genes were differentially expressed (FDR < 0.05) and Pcsk5 is a potential downstream effector of the Notch4 signaling pathway (P < 0.001). Low expression of Pcsk5 positively correlates with good survival in patients with PDAC (P = 0.028). We have identified a novel role for Notch4 signaling with tumor-promoting function in pancreatic tumorigenesis. Our study also uncovered a novel association between Pcsk5 and Notch4 signaling in PDAC. Significance: We demonstrated that global inactivation of Notch4 significantly improved the survival of an aggressive mouse model for PDAC and provided preclinical evidence that Notch4 and Pcsk5 are novel targets for PDAC therapies.

Propranolol Therapy in Infantile Hemangioma: It Is Not Just About the Beta.

BACKGROUND: Propranolol, a nonselective ß-adrenergic receptor antagonist, is approved by the U.S. Food and Drug Administration to treat problematic infantile hemangiomas, but a subset of patients experience treatment complications. Parents wary of long-term use and side effects consult plastic surgeons on surgical options or as a second opinion. Understanding the mechanism(s) of action of propranolol will allow plastic surgeons to better inform parents. METHODS: A systemic literature search was performed to query published translational and basic science studies on propranolol effects on infantile hemangiomas and cells derived from these lesions. RESULTS: In experimental studies, propranolol was antiproliferative and cytotoxic against hemangioma endothelial and stem cells and affected infantile hemangioma perivascular cell contractility. Propranolol inhibited migration, network formation, vascular endothelial growth factor A production, and vascular endothelial growth factor receptor 2 activation and down-regulated PI3K/AKT and mitogen-activated protein kinase signaling in hemangioma endothelial cells, but it increased ERK1/2 activity in hemangioma stem cells. At effective clinical doses, measured propranolol plasma concentration is 100 times higher than necessary for complete ß-adrenergic receptor blockade, yet was 10 to 100 times less than required to induce hemangioma stem cell death. CONCLUSIONS: Propranolol targets multiple cell types in infantile hemangiomas by means of ß-adrenergic receptor-dependent and -independent mechanisms. Plasma concentration played a significant role. At clinically relevant doses, incomplete infantile hemangioma suppression may explain the rebound phenomenon and worsening ulceration, and propranolol off target effects may lead to commonly reported adverse effects, such as sleep and gastrointestinal disturbances. Propranolol limitations and complications underscore the importance of surgical treatment options in cases of rebound and severe adverse effects. Surgical intervention remains an important treatment choice when parents are hesitant to use propranolol.