Ca2+/calmodulin kinase II-dependent regulation of ßIV-spectrin modulates cardiac fibroblast gene expression, proliferation, and contractility.

Fibrosis is a pronounced feature of heart disease and the result of dysregulated activation of resident cardiac fibroblasts (CFs). Recent work identified stress-induced degradation of the cytoskeletal protein ßIV-spectrin as an important step in CF activation and cardiac fibrosis. Furthermore, loss of ßIV-spectrin was found to depend on Ca2+/calmodulin-dependent kinase II (CaMKII). Therefore, we sought to determine the mechanism for CaMKII-dependent regulation of ßIV-spectrin and CF activity. Computational screening and MS revealed a critical serine residue (S2250 in mouse and S2254 in human) in ßIV-spectrin phosphorylated by CaMKII. Disruption of ßIV-spectrin/CaMKII interaction or alanine substitution of ßIV-spectrin Ser2250 (ßIV-S2254A) prevented CaMKII-induced degradation, whereas a phosphomimetic construct (ßIV-spectrin with glutamic acid substitution at serine 2254 [ßIV-S2254E]) showed accelerated degradation in the absence of CaMKII. To assess the physiological significance of this phosphorylation event, we expressed exogenous ßIV-S2254A and ßIV-S2254E constructs in ßIV-spectrin-deficient CFs, which have increased proliferation and fibrotic gene expression compared with WT CFs. ßIV-S2254A but not ßIV-S2254E normalized CF proliferation, gene expression, and contractility. Pathophysiological targeting of ßIV-spectrin phosphorylation and subsequent degradation was identified in CFs activated with the profibrotic ligand angiotensin II, resulting in increased proliferation and signal transducer and activation of transcription 3 nuclear accumulation. While therapeutic delivery of exogenous WT ßIV-spectrin partially reversed these trends, ßIV-S2254A completely negated increased CF proliferation and signal transducer and activation of transcription 3 translocation. Moreover, we observed ßIV-spectrin phosphorylation and associated loss in total protein within human heart tissue following heart failure. Together, these data illustrate a considerable role for the ßIV-spectrin/CaMKII interaction in activating profibrotic signaling.

Annexin A3 is necessary for parallel artery-vein alignment in the mouse retina.

BACKGROUND: Annexin A3 (Anxa3) is a member of the calcium-regulated, cell membrane-binding family of annexin proteins. We previously confirmed that Anxa3 is expressed in the endothelial lineage in vertebrates and that loss of anxa3 in Xenopus laevis leads to embryonic blood vessel defects. However, the biological function of Anxa3 in mammals is completely unknown. In order to investigate Anxa3 vascular function in mammals, we generated an endothelial cell-specific Anxa3 conditional knockout mouse model (Anxa3f/f ;Tie2-Cre). RESULTS: Anxa3f/f ;Tie2-Cre mice are born at Mendelian ratios and display morphologically normal blood vessels during development. However, loss of Anxa3 leads to artery-vein (AV) misalignment characterized by atypical AV crossovers in the postnatal and adult retina. CONCLUSIONS: Anxa3 is not essential for embryonic blood vessel formation but is required for proper parallel AV alignment in the murine retina. AV crossovers associated with Anxa3f/f ;Tie2-Cre mice are similar to AV intersections observed in patients with branch retinal vein occlusion (BRVO), although we did not observe occluded vessels. This new Anxa3 mouse model may provide a basis for understanding AV crossover formation associated with BRVO.

Vascular deficiency of Smad4 causes arteriovenous malformations: a mouse model of Hereditary Hemorrhagic Telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder that leads to abnormal connections between arteries and veins termed arteriovenous malformations (AVM). Mutations in TGFß pathway members ALK1, ENG and SMAD4 lead to HHT. However, a Smad4 mouse model of HHT does not currently exist. We aimed to create and characterize a Smad4 endothelial cell (EC)-specific, inducible knockout mouse (Smad4f/f;Cdh5-CreERT2) that could be used to study AVM development in HHT. We found that postnatal ablation of Smad4 caused various vascular defects, including the formation of distinct AVMs in the neonate retina. Our analyses demonstrated that increased EC proliferation and size, altered mural cell coverage and distorted artery-vein gene expression are associated with Smad4 deficiency in the vasculature. Furthermore, we show that depletion of Smad4 leads to decreased Vegfr2 expression, and concurrent loss of endothelial Smad4 and Vegfr2 in vivo leads to AVM enlargement. Our work provides a new model in which to study HHT-associated phenotypes and links the TGFß and VEGF signaling pathways in AVM pathogenesis.

CaMKII-dependent late Na+ current increases electrical dispersion and arrhythmia in ischemia-reperfusion.

The mechanisms underlying Ca2+/calmodulin-dependent protein kinase II (CaMKII)-induced arrhythmias in ischemia-reperfusion (I/R) are not fully understood. We tested the hypothesis that CaMKII increases late Na+ current ( INa,L) via phosphorylation of Nav1.5 at Ser571 during I/R, thereby increasing arrhythmia susceptibility. To test our hypothesis, we studied isolated, Langendorff-perfused hearts from wild-type (WT) mice and mice expressing Nav channel variants Nav1.5-Ser571E (S571E) and Nav1.5-Ser571A (S571A). WT hearts showed a significant increase in the levels of phosphorylated CaMKII and Nav1.5 at Ser571 [p-Nav1.5(S571)] after 15 min of global ischemia (just before the onset of reperfusion). Optical mapping experiments revealed an increase in action potential duration (APD) and APD dispersion without changes in conduction velocity during I/R in WT and S571E compared with S571A hearts. At the same time, WT and S571E hearts showed an increase in spontaneous arrhythmia events (e.g., premature ventricular contractions) and an increase in the inducibility of reentrant arrhythmias during reperfusion. Pretreatment of WT hearts with the Na+ channel blocker mexiletine (10 µM) normalized APD dispersion and reduced arrhythmia susceptibility during I/R. We conclude that CaMKII-dependent phosphorylation of Nav1.5 is a crucial driver for increased INa,L, arrhythmia triggers, and substrate during I/R. Selective targeting of this CaMKII-dependent pathway may have therapeutic potential for reducing arrhythmias in the setting of I/R. NEW & NOTEWORTHY Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of Nav1.5 at Ser571 leads to a prolongation of action potential duration (APD), increased APD dispersion, and increased arrhythmia susceptibility after ischemia-reperfusion in isolated mouse hearts. Genetic ablation of the CaMKII-dependent phosphorylation site Ser571 on Nav1.5 or low-dose mexiletine (to inhibit late Na+ current) reduced APD dispersion, arrhythmia triggers, and ventricular tachycardia inducibility.

Growth arrest in the ribosomopathy, Bowen-Conradi syndrome, is due to dramatically reduced cell proliferation and a defect in mitotic progression.

Bowen-Conradi syndrome (BCS) is a ribosomopathy characterized by severe developmental delay and growth failure that typically leads to death by one year of age. It is caused by a c.257A>G, p.D86G substitution in the ribosomal biogenesis protein, Essential for Mitotic Growth 1 (EMG1). We generated a knock-in of the D86G substitution in mice to characterize the effects of EMG1 deficiency, particularly in the brain, where EMG1 expression is high. Embryos homozygous for the mutation in Emg1 were small for gestational age with neural tube defects, and died between embryonic days 8.5 and 12.5. These embryos exhibited dramatically reduced cell proliferation, which we also detected in autopsy brain tissue and bone marrow of BCS patients, consistent with a requirement for high levels of EMG1 in tissues with rapid cell proliferation. In fibroblasts derived from the BCS mouse embryos, we detected a high proportion of binucleated cells, indicating that a mitotic defect underlies the growth arrest in BCS. These studies add to growing evidence of a link between ribosome biogenesis, mitotic progression, and brain development that is currently unexplored.

A Size-Selective Intracellular Delivery Platform.

Identifying and separating a subpopulation of cells from a heterogeneous mixture are essential elements of biological research. Current approaches require detailed knowledge of unique cell surface properties of the target cell population. A method is described that exploits size differences of cells to facilitate selective intracellular delivery using a high throughput microfluidic device. Cells traversing a constriction within this device undergo a transient disruption of the cell membrane that allows for cytoplasmic delivery of cargo. Unique constriction widths allow for optimization of delivery to cells of different sizes. For example, a 4 µm wide constriction is effective for delivery of cargo to primary human T-cells that have an average diameter of 6.7 µm. In contrast, a 6 or 7 µm wide constriction is best for large pancreatic cancer cell lines BxPc3 (10.8 µm) and PANC-1 (12.3 µm). These small differences in cell diameter are sufficient to allow for selective delivery of cargo to pancreatic cancer cells within a heterogeneous mixture containing T-cells. The application of this approach is demonstrated by selectively delivering dextran-conjugated fluorophores to circulating tumor cells in patient blood allowing for their subsequent isolation and genomic characterization.

Transcatheter device closure of perimembranous ventricular septal defect in children treated with prophylactic oral steroids: acute and mid-term results of a single-centre, prospective, observational study.

UNLABELLED: Background and Objective Although transcatheter closure of perimembranous ventricular septal defect is emerging as an accepted, viable alternative, conduction disturbances still remain a major concern. Although steroid treatment has shown encouraging results with complete recovery, efficacy of prophylactic use of steroids is still speculative. We aim to study the mid-term outcome of perimembranous ventricular septal defect closure in children who received prophylactic oral steroids. Materials and methods A prospective study was designed and antegrade device closure was attempted in eligible children who met the following inclusion criteria: age 3-18 years and weight >10 kg, defect diameter ⩽12 mm, and symptomatic, haemodynamic changes or history of infective endocarditis. Prophylactic steroid protocol consisted of 2 weeks oral prednisolone (1 mg/kg/day) initiated immediately after the procedure, and in the event of bradyarrhythmia it was escalated to 2 mg/kg. Patients were regularly followed-up at 1, 6, and 12 months and then annually. Patients with post-procedure heart block underwent Holter monitoring after a minimum of 1 year interval. RESULTS: Between May, 2007 and August, 2012, successful device closure was accomplished in 290/297 patients. Mean age and weight were 9±3.12 years and 21±8.27 kg, respectively. The defect measured 5±1.38 mm on echocardiography. Mean fluoroscopy time was 12.98±8.64 minutes. Eight patients with major complications included one each with device embolisation, haemolysis, severe aortic regurgitation, and five with bradyarrhythmias, including complete atrioventricular block in three, Mobitz II in one, and bifascicular block in one. Patients with complete atrioventricular block responded to high-dose steroid and temporary pacemaker. Minor complications included post-procedure heart block (n=22) and blood loss (n=2). At 18.23±13.15 months follow-up, 8/27 (five major, 22 minor) with arrhythmia had persistent post-procedure heart block of no clinical consequences. CONCLUSION: In our patient population, transcatheter device closure of the perimembranous ventricular septal defect with prophylactic oral steroid resulted in excellent closure rate and acceptably low incidence of conduction disturbances at mid-term follow-up.

De-repression of FOXO3a death axis by microRNA-132 and -212 causes neuronal apoptosis in Alzheimer's disease.

Alzheimer's disease (AD) is a multifactorial and fatal neurodegenerative disorder for which the mechanisms leading to profound neuronal loss are incompletely recognized. MicroRNAs (miRNAs) are recently discovered small regulatory RNA molecules that repress gene expression and are increasingly acknowledged as prime regulators involved in human brain pathologies. Here we identified two homologous miRNAs, miR-132 and miR-212, downregulated in temporal cortical areas and CA1 hippocampal neurons of human AD brains. Sequence-specific inhibition of miR-132 and miR-212 induces apoptosis in cultured primary neurons, whereas their overexpression is neuroprotective against oxidative stress. Using primary neurons and PC12 cells, we demonstrate that miR-132/212 controls cell survival by direct regulation of PTEN, FOXO3a and P300, which are all key elements of AKT signaling pathway. Silencing of these three target genes by RNAi abrogates apoptosis caused by the miR-132/212 inhibition. We further demonstrate that mRNA and protein levels of PTEN, FOXO3a, P300 and most of the direct pro-apoptotic transcriptional targets of FOXO3a are significantly elevated in human AD brains. These results indicate that the miR-132/miR-212/PTEN/FOXO3a signaling pathway contributes to AD neurodegeneration.

Outbreak of salmonellosis linked to live poultry from a mail-order hatchery.

BACKGROUND: Outbreaks of human salmonella infections are increasingly associated with contact with live poultry, but effective control measures are elusive. In 2005, a cluster of human salmonella Montevideo infections with a rare pattern on pulsed-field gel electrophoresis (the outbreak strain) was identified by PulseNet, a national subtyping network. METHODS: In cooperation with public health and animal health agencies, we conducted multistate investigations involving patient interviews, trace-back investigations, and environmental testing at a mail-order hatchery linked to the outbreak in order to identify the source of infections and prevent additional illnesses. A case was defined as an infection with the outbreak strain between 2004 and 2011. RESULTS: From 2004 through 2011, we identified 316 cases in 43 states. The median age of the patient was 4 years. Interviews were completed with 156 patients (or their caretakers) (49%), and 36 of these patients (23%) were hospitalized. Among the 145 patients for whom information was available, 80 (55%) had bloody diarrhea. Information on contact with live young poultry was available for 159 patients, and 122 of these patients (77%) reported having such contact. A mail-order hatchery in the western United States was identified in 81% of the trace-back investigations, and the outbreak strain was isolated from samples collected at the hatchery. After interventions at the hatchery, the number of human infections declined, but transmission continued. CONCLUSIONS: We identified a prolonged multistate outbreak of salmonellosis, predominantly affecting young children and associated with contact with live young poultry from a mail-order hatchery. Interventions performed at the hatchery reduced, but did not eliminate, associated human infections, demonstrating the difficulty of eliminating salmonella transmission from live poultry.

Clinical and angiographic follow-up of coronary artery fistula interventions in children: techniques and classification revisited.

BACKGROUND: Transcatheter closure of coronary artery fistula has emerged as a safe and effective alternative to surgery. However, follow-up angiographic data after closure of the coronary artery fistula is extremely limited. We report our clinical and angiographic follow-up of children who underwent either transcatheter or surgical closure. METHOD: Clinical profile, echocardiography parameters, and closure technique were retrospectively reviewed from the hospital charts. Since 2007, 15 children have been intervened and followed up with electrocardiography, echocardiography, and angiography. RESULTS: A total of 15 children (six girls), with mean age of 6.7±5.4 years and weighing 16.3±9.8 kg, underwent successful closure (transcatheter=13, surgical=2) without periprocedural complication. Coronary artery fistula arose from the right (n=7) and left coronary artery (n=8) and drained into the right atrium or the right ventricle. Transcatheter closure was carried out using a duct occluder. Of the patients, two underwent surgical closure of the fistula on a beating heart. At 31.8±18.7 months follow-up, all the children were asymptomatic and had no evidence of myocardial ischaemia or infarction. However, follow-up angiography revealed thrombotic occlusion of fistula with the patent parent coronary artery in those having branch coronary artery fistula, and five of seven patients with parent coronary artery fistula had near-complete occlusion of fistula extending into the native coronary artery. CONCLUSION: Follow-up angiography revealed a high incidence of parent artery occlusion when the fistula was arising from the native artery and not from one of its branches. Coronary artery fistula intervention of the parent coronary artery fistula always carries the potential risk of ischaemia, unless the distal-most exiting segment is the primary site of occlusion.

Endothelial Zmiz1 modulates physiological and pathophysiological angiogenesis during retinal development.

Angiogenesis is a highly coordinated process involving the control of various endothelial cell behaviors. Mechanisms for transcription factor involvement in the regulation of endothelial cell dynamics and angiogenesis have become better understood, however much remains unknown, especially the role of non-DNA binding transcriptional cofactors. Here, we show that Zmiz1, a transcription cofactor, is enriched in the endothelium and critical for embryonic vascular development, postnatal retinal angiogenesis, and pathological angiogenesis in oxygen induced retinopathy (OIR). In mice, endothelial cell-specific deletion of Zmiz1 during embryogenesis led to lethality due to abnormal angiogenesis and vascular defects. Inducible endothelial cell-specific ablation of Zmiz1 postnatally resulted in impaired retinal vascular outgrowth, decreased vascular density, and increased vessel regression. In addition, angiogenic sprouting in the superficial and deep layers of the retina was markedly reduced. Correspondingly, vascular sprouting in fibrin bead assays was significantly reduced in the absence of Zmiz1, while further in vitro and in vivo evidence also suggested deficits in EC migration. In agreement with the defective sprouting angiogenesis phenotype, gene expression analysis of isolated retinal endothelial cells revealed downregulation of tip-cell enriched genes upon inactivation of Zmiz1. Lastly, our study suggested that endothelial Zmiz1 is critical for intraretinal revascularization following hypoxia exposure in the OIR model. Taken together, these findings begin to define the previously unspecified role of endothelial Zmiz1 in physiological and pathological angiogenesis.

Zmiz1 is a novel regulator of lymphatic endothelial cell gene expression and function.

Zinc Finger MIZ-Type Containing 1 (Zmiz1), also known as ZIMP10 or RAI17, is a transcription cofactor and member of the Protein Inhibitor of Activated STAT (PIAS) family of proteins. Zmiz1 is critical for a variety of biological processes including vascular development. However, its role in the lymphatic vasculature is unknown. In this study, we utilized human dermal lymphatic endothelial cells (HDLECs) and an inducible, lymphatic endothelial cell (LEC)-specific Zmiz1 knockout mouse model to investigate the role of Zmiz1 in LECs. Transcriptional profiling of ZMIZ1-deficient HDLECs revealed downregulation of genes crucial for lymphatic vessel development. Additionally, our findings demonstrated that loss of Zmiz1 results in reduced expression of proliferation and migration genes in HDLECs and reduced proliferation and migration in vitro. We also presented evidence that Zmiz1 regulates Prox1 expression in vitro and in vivo by modulating chromatin accessibility at Prox1 regulatory regions. Furthermore, we observed that loss of Zmiz1 in mesenteric lymphatic vessels significantly reduced valve density. Collectively, our results highlight a novel role of Zmiz1 in LECs and as a transcriptional regulator of Prox1, shedding light on a previously unknown regulatory factor in lymphatic vascular biology.

Loss of Zmiz1 in mice leads to impaired cortical development and autistic-like behaviors.

De novo mutations in transcriptional regulators are emerging as key risk factors contributing to the etiology of neurodevelopmental disorders. Human genetic studies have recently identified ZMIZ1 and its de novo mutations as causal of a neurodevelopmental syndrome strongly associated with intellectual disability, autism, ADHD, microcephaly, and other developmental anomalies. However, the role of ZMIZ in brain development or how ZMIZ1 mutations cause neurological phenotypes is unknown. Here, we generated a forebrain-specific Zmiz1 mutant mouse model that develops brain abnormalities, including cortical microcephaly, corpus callosum dysgenesis, and abnormal differentiation of upper-layer cortical neurons. Behaviorally, Zmiz1 mutant mice show alterations in motor activity, anxiety, communication, and social interactions with strong sex differences, resembling phenotypes associated with autism. Molecularly, Zmiz1 deficiency leads to transcriptomic changes disrupting neurogenesis, neuron differentiation programs, and synaptic signaling. We identified Zmiz1-mediated downstream regulation of key neurodevelopmental factors, including Lhx2, Auts2, and EfnB2. Importantly, reactivation of the EfnB2 pathway by exogenous EFNB2 recombinant protein rescues the dendritic outgrowth deficits in Zmiz1 mutant cortical neurons. Overall, our in vivo findings provide insight into Zmiz1 function in cortical development and reveal mechanistic underpinnings of ZMIZ1 syndrome, thereby providing valuable information relevant to future studies on this neurodevelopmental disorder.

An Adolescent Girl with Acute-on-Chronic Abdominal Pain.

We present an 11-year-old pediatric patient with acute-on-chronic abdominal pain found to have a large intra-abdominal abscess with a concomitant dermoid cyst. Acute-on-chronic abdominal pain has one of the broadest and, in our case, ever-changing differential diagnoses. Exploratory laparoscopy revealed a severe pelvic inflammatory process with a large abscess and extensive omental and bowel adhesions, a left ovarian cyst, a shortened appendix with thickened tip, and purulent fluid in the cul-de-sac. These findings suggested a ruptured appendix leading to a large abscess with adjacent ovarian dermoid cyst, and an appendectomy was performed. Our patient responded well to continued intravenous antibiotics, and her drain was removed on the day of discharge. She was sent home with an additional 2 weeks of oral cefdinir and metronidazole. Follow-up ultrasound showed dramatic cyst resolution, and no further intervention was needed. [Pediatr Ann. 2023;52(1):e36-e38.].

COVID-19-Induced Encephalopathy: A Case Report.

While COVID-19 is known to cause common neurological manifestations such as loss of taste and smell, headaches, and myalgias, rare and severe neurological complications can also occur. We describe the hospitalization of a middle-aged Caucasian woman who presented with altered mental status and an absence of moderate-severe pulmonary symptoms. The patient tested positive for COVID-19 and experienced a tonic-clonic seizure six days after admission. Diagnostic testing, including cerebrospinal fluid analysis, blood cultures, urine cultures, brain imaging, and electroencephalograms were unremarkable, indicating a global encephalopathic state. This case highlights the need for clinicians to anticipate neurological complications when managing patients with COVID-19, especially when respiratory symptoms are minimal or absent. Moreover, further research on COVID-19-induced encephalopathy is crucial to improve patient outcomes and inform clinical practice.

Role of Temporomandibular Total Joint Replacement in Correction of Facial Deformities.

Introduction: Individuals suffering from facial deformities experience esthetic, functional and social setback in their lives every day. These patients benefit greatly with single-stage temporomandibular joint replacement along with orthognathic surgical procedures to correct functional as well as esthetic components. Methodology: Five individuals with facial deformities due to hemifacial macrosomia, fibrous dysplasia, idiopathic condylar resorption, ankylosis, etc., were treated with total joint replacement along with orthognathic surgical procedures for functional and esthetic correction. The customized temporomandibular joint was digitally custom designed, and the orthognathic procedures were virtually planned on NemoFAB software before performing the surgical procedure. Results: Intraoperatively, the overall time was reduced considerably with minimal unpredictable complications. One-week postoperative computed tomographs were obtained and superimposed on preoperative virtual surgical planning which showed minimal discrepancy. Follow-up period of 18-30 months was maintained for all the patients with stable results, minimal relapse and satisfactory functional abilities. Discussion: Total joint replacement along with orthognathic procedure provides single-stage functional and esthetic corrections for the patients, considerably improving their quality of life. Precise preoperative surgical planning proves to be an indispensable step for achieving desired results with minimal margin of error and avoid any unseen complications during the surgical procedure.

A Novel Method to Rehabilitate Post-mucormycosis Maxillectomy Defect by Using Patient-Specific Zygoma Implant.

Aim: During second wave of COVID pandemic, India faced heavy surge of mucormycosis. Treatment option for these patients included either total or partial maxillectomy with primary closure. Rehabilitation of these patients became challenging because of their age and size of defect. The purpose of the present study is to present a new digital technique for the fabrication of patient-specific zygoma implants (PSI) and to report on its survival and complication rates. Material and Methods: Total 21 patients who had undergone either partial or total maxillectomy after mucormycosis and who were disease-free clinically and radiographically for 6 or more months post-resection were rehabilitated using patient-specific zygoma implant. CT scan was obtained for all patients post-maxillectomy for evaluation of existing bone condition. Exocad software was used for virtual surgical planning of zygoma implant considering surgical and prosthetic technicality to achieve goal of maximum functionality and sustainability. Result: All the patients were followed up after 15, 30, 45 and 90 days and there after every month for evaluation of soft tissue healing, infection, dehiscence, loosening of prosthesis, eating efficiency and aesthetic. Follow-up period for all 15 patients was in the range of 6-12 months. Conclusion: In case of post-mucor maxillectomy patients, use of PSI offers the advantages of minimal bone augmentation, reduction in time required to restore lost function, and reduced financial burden of multiple procedures. Therefore, PSI may represent a valid alternative treatment for the prosthetic restoration of post-mucor maxillectomy patients.

Spectrin-Based Regulation of Cardiac Fibroblast Cell-Cell Communication.

Cardiac fibroblasts (CFs) maintain the fibrous extracellular matrix (ECM) that supports proper cardiac function. Cardiac injury induces a transition in the activity of CFs to promote cardiac fibrosis. CFs play a critical role in sensing local injury signals and coordinating the organ level response through paracrine communication to distal cells. However, the mechanisms by which CFs engage cell-cell communication networks in response to stress remain unknown. We tested a role for the action-associated cytoskeletal protein ßIV-spectrin in regulating CF paracrine signaling. Conditioned culture media (CCM) was collected from WT and ßIV-spectrin deficient (qv4J) CFs. WT CFs treated with qv4J CCM showed increased proliferation and collagen gel compaction compared to control. Consistent with the functional measurements, qv4J CCM contained higher levels of pro-inflammatory and pro-fibrotic cytokines and increased concentration of small extracellular vesicles (30-150 nm diameter, exosomes). Treatment of WT CFs with exosomes isolated from qv4J CCM induced a similar phenotypic change as that observed with complete CCM. Treatment of qv4J CFs with an inhibitor of the ßIV-spectrin-associated transcription factor, STAT3, decreased the levels of both cytokines and exosomes in conditioned media. This study expands the role of the ßIV-spectrin/STAT3 complex in stress-induced regulation of CF paracrine signaling.

Zmiz1 is a novel regulator of lymphatic endothelial cell gene expression and function.

Zinc Finger MIZ-Type Containing 1 (Zmiz1), also known as ZIMP10 or RAI17, is a transcription cofactor and member of the Protein Inhibitor of Activated STAT (PIAS) family of proteins. Zmiz1 is critical for a variety of biological processes including vascular development. However, its role in the lymphatic vasculature is unknown. In this study, we utilized human dermal lymphatic endothelial cells (HDLECs) and an inducible, lymphatic endothelial cell (LEC)-specific Zmiz1 knockout mouse model to investigate the role of Zmiz1 in LECs. Transcriptional profiling of Zmiz1-deficient HDLECs revealed downregulation of genes crucial for lymphatic vessel development. Additionally, our findings demonstrated that loss of Zmiz1 results in reduced expression of proliferation and migration genes in HDLECs and reduced proliferation and migration in vitro. We also presented evidence that Zmiz1 regulates Prox1 expression in vitro and in vivo by modulating chromatin accessibility at Prox1 regulatory regions. Furthermore, we observed that loss of Zmiz1 in mesenteric lymphatic vessels significantly reduced valve density. Collectively, our results highlight a novel role of Zmiz1 in LECs and as a transcriptional regulator of Prox1, shedding light on a previously unknown regulatory factor in lymphatic vascular biology.