ADAMTSL2 mutations determine the phenotypic severity in geleophysic dysplasia.

Geleophysic dysplasia-1 (GD1) is an autosomal recessive disorder caused by ADAMTS-like 2 (ADAMTSL2) variants. It is characterized by distinctive facial features, limited joint mobility, short stature, brachydactyly, and life-threatening cardiorespiratory complications. The clinical spectrum spans from perinatal lethality to milder adult phenotypes. We developed and characterized cellular and mouse models, to replicate the genetic profile of a patient who is compound heterozygous for 2 ADAMTSL2 variants, namely p.R61H and p.A165T. The impairment of ADAMTSL2 secretion was observed in both variants, but p.A165T exhibited a more severe impact. Mice carrying different allelic combinations revealed a spectrum of phenotypic severity, from lethality in knockout homozygotes to mild growth impairment observed in adult p.R61H homozygotes. Homozygous and hemizygous p.A165T mice survived but displayed severe respiratory and cardiac dysfunction. The respiratory dysfunction mainly affected the expiration phase, and some of these animals had microscopic post-obstructive pneumonia. Echocardiograms and MRI studies revealed a significant systolic dysfunction, accompanied by a reduction of the aortic root size. Histology verified the presence of hypertrophic cardiomyopathy with myocyte hypertrophy, chondroid metaplasia, and mild interstitial fibrosis. This study revealed a substantial correlation between the degree of impaired ADAMTSL2 secretion and the severity of the observed phenotype in GD1.

EFEMP1 haploinsufficiency causes a Marfan-like hereditary connective tissue disorder.

Phenotypic features of a hereditary connective tissue disorder, including craniofacial characteristics, hyperextensible skin, joint laxity, kyphoscoliosis, arachnodactyly, inguinal hernia, and diverticulosis associated with biallelic pathogenic variants in EFEMP1 have been previously described in four patients. Genome sequencing on a proband and her mother with comparable phenotypic features revealed that both patients were heterozygous for a stop-gain variant c.1084C>T (p.Arg362*). Complementary RNA-seq on fibroblasts revealed significantly reduced levels of mutant EFEMP1 transcript. Considering the absence of other molecular explanations, we extrapolated that EFEMP1 could be the cause of the patient's phenotypes. Furthermore, nonsense-mediated decay was demonstrated for the mutant allele as the principal mechanism for decreased levels of EFEMP1 mRNA. We provide strong clinical and genetic evidence for the haploinsufficiency of EFEMP1 due to nonsense-medicated decay to cause severe kyphoscoliosis, generalized hypermobility of joints, high and narrow arched palate, and potentially severe diverticulosis. To the best of our knowledge, this is the first report of an autosomal dominant EFEMP1-associated hereditary connective tissue disorder and therefore expands the phenotypic spectrum of EFEMP1 related disorders.

c-Myc Drives inflammation of the maternal-fetal interface, and neonatal lung remodeling induced by intra-amniotic inflammation.

Background: Intra-amniotic inflammation (IAI) is associated with increased risk of preterm birth and bronchopulmonary dysplasia (BPD), but the mechanisms by which IAI leads to preterm birth and BPD are poorly understood, and there are no effective therapies for preterm birth and BPD. The transcription factor c-Myc regulates various biological processes like cell growth, apoptosis, and inflammation. We hypothesized that c-Myc modulates inflammation at the maternal-fetal interface, and neonatal lung remodeling. The objectives of our study were 1) to determine the kinetics of c-Myc in the placenta, fetal membranes and neonatal lungs exposed to IAI, and 2) to determine the role of c-Myc in modulating inflammation at the maternal-fetal interface, and neonatal lung remodeling induced by IAI. Methods: Pregnant Sprague-Dawley rats were randomized into three groups: 1) Intra-amniotic saline injections only (control), 2) Intra-amniotic lipopolysaccharide (LPS) injections only, and 3) Intra-amniotic LPS injections with c-Myc inhibitor 10058-F4. c-Myc expression, markers of inflammation, angiogenesis, immunohistochemistry, and transcriptomic analyses were performed on placenta and fetal membranes, and neonatal lungs to determine kinetics of c-Myc expression in response to IAI, and effects of prenatal systemic c-Myc inhibition on lung remodeling at postnatal day 14. Results: c-Myc was upregulated in the placenta, fetal membranes, and neonatal lungs exposed to IAI. IAI caused neutrophil infiltration and neutrophil extracellular trap (NET) formation in the placenta and fetal membranes, and neonatal lung remodeling with pulmonary hypertension consistent with a BPD phenotype. Prenatal inhibition of c-Myc with 10058-F4 in IAI decreased neutrophil infiltration and NET formation, and improved neonatal lung remodeling induced by LPS, with improved alveolarization, increased angiogenesis, and decreased pulmonary vascular remodeling. Discussion: In a rat model of IAI, c-Myc regulates neutrophil recruitment and NET formation in the placenta and fetal membranes. c-Myc also participates in neonatal lung remodeling induced by IAI. Further studies are needed to investigate c-Myc as a potential therapeutic target for IAI and IAI-associated BPD.

Comparative Profiling of TG2 and Its Effectors in Human Relapsing Remitting and Progressive Multiple Sclerosis.

Multiple Sclerosis (MS) is a chronic CNS autoimmune disease characterized by immune-mediated demyelination, axon loss, and disability. Dysregulation of transglutaminase-2 (TG2) has been implicated in disease initiation and progression. Herein, TG2 expression in post-mortem human brain tissue from Relapsing Remitting MS (RRMS) or Progressive MS (PMS) individuals were examined and correlated with the presence of TG2 binding partners and effectors implicated in the processes of inflammation, scar formation, and the antagonism of repair. Tissues from Relapsing-Remitting Multiple Sclerosis (RRMS; n = 6), Progressive Multiple Sclerosis (PMS; n = 5), and non-MS control (n = 6) patients underwent immunohistochemistry for TG2, PLA2, COX-2, FN, CSPG, and HSPG. TG2 was strongly upregulated in active RRMS and PMS lesions, within blood vessels and the perivascular tissue of sclerotic plaques. TG2 colocalization was observed with GFAP+ astrocytes and ECM, including FN, HSPG, and CSPG, which also increased in either RRMS or PMS lesions. Although TG2 was not colocalized with inflammatory mediators COX-2 and PLA2, or the macrophage-microglia marker Iba1, its increased expression correlated with their elevation in active RRMS and PMS lesions. In summary, the correlation of strong TG2 induction in either RRMS or PMS with some of its binding partners but not others implicates potentially different roles for TG2 in disparate MS forms that may warrant further investigation.

Endothelial c-Myc knockout enhances diet-induced liver inflammation and fibrosis.

Endothelial cells play an essential role in inflammation through synthesis and secretion of chemoattractant cytokines and expression of adhesion molecules required for inflammatory cell attachment and infiltration. The mechanisms by which endothelial cells control the pro-inflammatory response depend on the type of inflammatory stimuli, endothelial cell origin, and tissue involved. In the present study, we investigated the role of the transcription factor c-Myc in inflammation using a conditional knockout mouse model in which Myc is specifically deleted in the endothelium. At a systemic level, circulating monocytes, the chemokine CCL7, and the extracellular-matrix protein osteopontin were significantly increased in endothelial c-Myc knockout (EC-Myc KO) mice, whereas the cytokine TNFSF11 was downregulated. Using an experimental model of steatohepatitis, we investigated the involvement of endothelial c-Myc in diet-induced inflammation. EC-Myc KO animals displayed enhanced pro-inflammatory response, characterized by increased expression of pro-inflammatory cytokines and leukocyte infiltration, and worsened liver fibrosis. Transcriptome analysis identified enhanced expression of genes associated with inflammation, fibrosis, and hepatocellular carcinoma in EC-Myc KO mice relative to control (CT) animals after short-exposure to high-fat diet. Analysis of a single-cell RNA-sequencing dataset of human cirrhotic livers indicated downregulation of MYC in endothelial cells relative to healthy controls. In summary, our results suggest a protective role of endothelial c-Myc in diet-induced liver inflammation and fibrosis. Targeting c-Myc and its downstream pathways in the endothelium may constitute a potential strategy for the treatment of inflammatory disease.

Engineering polysialic acid on Schwann cells using polysialyltransferase gene transfer or purified enzyme exposure for spinal cord injury transplantation.

Polysialic acid (PolySia) is a critical post-translational modification on the neural cell adhesion molecule (NCAM, a.k.a., CD56), important for cell migration and axon growth during nervous system development, plasticity and repair. PolySia induction on Schwann cells (SCs) enhances their migration, axon growth support and ability to improve functional recovery after spinal cord injury (SCI) transplantation. In the current investigation two methods of PolySia induction on SCs, lentiviral vector transduction of the mouse polysialytransferase gene ST8SIA4 (LV-PST) or enzymatic engineering with a recombinant bacterial PST (PSTNm), were examined comparatively for their effects on PolySia induction, SC migration, the innate immune response and axon growth after acute SCI. PSTNm produced significant PolySia induction and a greater diversity of surface molecule polysialylation on SCs as evidenced by immunoblot. In the scratch wound assay, PSTNm was superior to LV-PST in the promotion of SC migration and gap closure. At 24 h after SCI transplantation, PolySia induction on SCs was most pronounced with LV-PST. Co-delivery of PSTNm with SCs, but not transient cell exposure, led to broader induction of PolySia within the injured spinal cord due to polysialylation upon both host cells and transplanted SCs. The innate immune response after SCI, measured by CD68 immunoreactivity, was similar among PolySia induction methods. LV-PST or PSTNm co-delivery with SCs provided a similar enhancement of SC migration and axon growth support above that of unmodified SCs. These studies demonstrate that LV-PST and PSTNm provide comparable acute effects on SC polysialation, the immune response and neurorepair after SCI.

The Comparative Utility of Viromer RED and Lipofectamine for Transient Gene Introduction into Glial Cells.

The introduction of genes into glial cells for mechanistic studies of cell function and as a therapeutic for gene delivery is an expanding field. Though viral vector based systems do exhibit good delivery efficiency and long-term production of the transgene, the need for transient gene expression, broad and rapid gene setup methodologies, and safety concerns regarding in vivo application still incentivize research into the use of nonviral gene delivery methods. In the current study, aviral gene delivery vectors based upon cationic lipid (Lipofectamine 3000) lipoplex or polyethylenimine (Viromer RED) polyplex technologies were examined in cell lines and primary glial cells for their transfection efficiencies, gene expression levels, and toxicity. The transfection efficiencies of polyplex and lipoplex agents were found to be comparable in a limited, yet similar, transfection setting, with or without serum across a number of cell types. However, differential effects on cell-specific transgene expression and reduced viability with cargo loaded polyplex were observed. Overall, our data suggests that polyplex technology could perform comparably to the market dominant lipoplex technology in transfecting various cells lines including glial cells but also stress a need for further refinement of polyplex reagents to minimize their effects on cell viability.

Perforin-2 is essential for intracellular defense of parenchymal cells and phagocytes against pathogenic bacteria.

Perforin-2 (MPEG1) is a pore-forming, antibacterial protein with broad-spectrum activity. Perforin-2 is expressed constitutively in phagocytes and inducibly in parenchymal, tissue-forming cells. In vitro, Perforin-2 prevents the intracellular replication and proliferation of bacterial pathogens in these cells. Perforin-2 knockout mice are unable to control the systemic dissemination of methicillin-resistant Staphylococcus aureus (MRSA) or Salmonella typhimurium and perish shortly after epicutaneous or orogastric infection respectively. In contrast, Perforin-2-sufficient littermates clear the infection. Perforin-2 is a transmembrane protein of cytosolic vesicles -derived from multiple organelles- that translocate to and fuse with bacterium containing vesicles. Subsequently, Perforin-2 polymerizes and forms large clusters of 100 Å pores in the bacterial surface with Perforin-2 cleavage products present in bacteria. Perforin-2 is also required for the bactericidal activity of reactive oxygen and nitrogen species and hydrolytic enzymes. Perforin-2 constitutes a novel and apparently essential bactericidal effector molecule of the innate immune system.

STING recognition of cytoplasmic DNA instigates cellular defense.

How the cell recognizes cytosolic DNA including DNA-based microbes to trigger host-defense-related gene activation remains to be fully resolved. Here, we demonstrate that STING (stimulator of interferon genes), an endoplasmic reticulum translocon-associated transmembrane protein, acts to detect cytoplasmic DNA species. STING homodimers were able to complex with self- (apoptotic, necrotic) or pathogen-related ssDNA and dsDNA and were indispensible for HSV-1-mediated transcriptional activation of a wide array of innate immune and proinflammatory genes in addition to type I IFN. Our data indicate that STING instigates cytoplasmic DNA-mediated cellular defense gene transcription and facilitates adoptive responses that are required for protection of the host. In contrast, chronic STING activation may manifest inflammatory responses and possibly autoimmune disease triggered by self-DNA.

Alterations in glutathione levels and apoptotic regulators are associated with acquisition of arsenic trioxide resistance in multiple myeloma.

Arsenic trioxide (ATO) has been tested in relapsed/refractory multiple myeloma with limited success. In order to better understand drug mechanism and resistance pathways in myeloma we generated an ATO-resistant cell line, 8226/S-ATOR05, with an IC50 that is 2-3-fold higher than control cell lines and significantly higher than clinically achievable concentrations. Interestingly we found two parallel pathways governing resistance to ATO in 8226/S-ATOR05, and the relevance of these pathways appears to be linked to the concentration of ATO used. We found changes in the expression of Bcl-2 family proteins Bfl-1 and Noxa as well as an increase in cellular glutathione (GSH) levels. At low, clinically achievable concentrations, resistance was primarily associated with an increase in expression of the anti-apoptotic protein Bfl-1 and a decrease in expression of the pro-apoptotic protein Noxa. However, as the concentration of ATO increased, elevated levels of intracellular GSH in 8226/S-ATOR05 became the primary mechanism of ATO resistance. Removal of arsenic selection resulted in a loss of the resistance phenotype, with cells becoming sensitive to high concentrations of ATO within 7 days following drug removal, indicating changes associated with high level resistance (elevated GSH) are dependent upon the presence of arsenic. Conversely, not until 50 days without arsenic did cells once again become sensitive to clinically relevant doses of ATO, coinciding with a decrease in the expression of Bfl-1. In addition we found cross-resistance to melphalan and doxorubicin in 8226/S-ATOR05, suggesting ATO-resistance pathways may also be involved in resistance to other chemotherapeutic agents used in the treatment of multiple myeloma.

Distribution of Bim determines Mcl-1 dependence or codependence with Bcl-xL/Bcl-2 in Mcl-1-expressing myeloma cells.

Dependence on Bcl-2 proteins is a common feature of cancer cells and provides a therapeutic opportunity. ABT-737 is an antagonist of antiapoptotic Bcl-2 proteins and therefore is a good predictor of Bcl-x(L)/Bcl-2 dependence. Surprisingly, analysis of Mcl-1-dependent multiple myeloma cell lines revealed codependence on Bcl-2/Bcl-x(L) in half the cells tested. Codependence is not predicted by the expression level of antiapoptotic proteins, rather through interactions with Bim. Consistent with these findings, acquired resistance to ABT-737 results in loss of codependence through redistribution of Bim to Mcl-1. Overall, these results suggest that complex interactions, and not simply expression patterns of Bcl-2 proteins, need to be investigated to understand Bcl-2 dependence and how to better use agents, such as ABT-737.

Darinaparsin induces a unique cellular response and is active in an arsenic trioxide-resistant myeloma cell line.

Here, we report on the organic arsenical darinaparsin (ZIO-101, S-dimethylarsino-glutathione) and its anti-myeloma activity compared with inorganic arsenic trioxide. Darinaparsin induced apoptosis in multiple myeloma cell lines in a dose-dependent manner, and the addition of N-acetylcysteine, which increases intracellular glutathione (GSH), blocked cytotoxicity of both darinaparsin and arsenic trioxide. In contrast to arsenic trioxide, intracellular GSH does not appear to be important for darinaparsin metabolism, as an inhibitor of GSH synthesis, buthionine sulfoximine, had little effect on drug activity. This discrepancy was resolved when we determined the effects of thiols on drug uptake. The addition of exogenous GSH, L-cysteine, or D-cysteine prevented darinaparsin cellular uptake and cell death but had no effect on the uptake or activity of arsenic trioxide, suggesting a difference in the transport mechanism of these two drugs. In addition, gene expression profiling revealed differences in the signaling of protective responses between darinaparsin and arsenic trioxide. Although both arsenicals induced a transient heat shock response, only arsenic trioxide treatment induced transcription of metal response genes and anti-oxidant genes related to the Nrf2-Keap1 pathway. In contrast to the protective responses, both arsenicals induced up-regulation of BH3-only proteins. Moreover, silencing of BH3-only proteins Noxa, Bim, and Bmf protected myeloma cells from darinaparsin-induced cell death. Finally, treatment of an arsenic trioxide-resistant myeloma cell line with darinaparsin resulted in dose-dependent apoptosis, indicating that cross-resistance does not necessarily develop between these two forms of arsenic in multiple myeloma cell lines. These results suggest darinaparsin may be useful as an alternative treatment in arsenic trioxide-resistant hematologic cancers.

Reactive oxygen species are not required for an arsenic trioxide-induced antioxidant response or apoptosis.

Arsenicals are both environmental carcinogens as well as therapeutic agents for the treatment of trypanosomiasis and more recently cancer. Arsenic trioxide (ATO) has been successfully used for the treatment of acute promyelocytic leukemia (APL) and has activity in multiple myeloma (MM). While signaling events associated with carcinogenesis have been well studied, it still remains to be determined which of these events are involved in anti-cancer signaling. To better define this response, gene expression profiling following ATO treatment of four MM cell lines was performed. The pattern was consistent with a strong antioxidative response, particularly of genes activated by Nrf2. While Nrf2 is expressed constitutively at the mRNA level, the protein is not detected in untreated cells. Consistent with inactivation of Keap1, Nrf2 protein is stabilized and present in the nucleus within 6 h of ATO treatment. Despite the activation of this antioxidative response, ROS may not be important in ATO-induced death. Inhibition of ATO-induced ROS with butylated hydroxyanisole (BHA) does not affect Nrf2 activation or cell death. Moreover, silencing Nrf2 had no effect on ATO-induced apoptosis. Together these data suggest that ROS is not important in the induction of the antioxidative response or cellular death by ATO.

BH3-only proteins Noxa, Bmf, and Bim are necessary for arsenic trioxide-induced cell death in myeloma.

The use of arsenic trioxide (ATO) to treat multiple myeloma (MM) is supported by preclinical studies as well as several phase 2 studies, but the precise mechanism(s) of action of ATO has not been completely elucidated. We used gene expression profiling to determine the regulation of apoptosis-related genes by ATO in 4 MM cell lines and then focused on Bcl-2 family genes. ATO induced up-regulation of 3 proapoptotic BH3-only proteins (Noxa, Bmf, and Puma) and down-regulation of 2 antiapoptotic proteins Mcl-1 and Bcl-X(L). Coimmunoprecipitation demonstrated that Noxa and Puma bind Mcl-1 to release Bak and Bim within 6 hours of ATO addition. Bak and Bim are also released from Bcl-X(L). Silencing of Bmf, Noxa, and Bim significantly protected cells from ATO-induced apoptosis, while Puma silencing had no effect. Consistent with a role for Noxa inhibition of Mcl-1, the Bad-mimetic ABT-737 synergized with ATO in the killing of 2 MM lines. Finally, Noxa expression was enhanced by GSH depletion and inhibited by increasing GSH levels in the cells. Understanding the pattern of BH3-only protein response should aid in the rational design of arsenic-containing regimens.

High expression of bfl-1 contributes to the apoptosis resistant phenotype in B-cell chronic lymphocytic leukemia.

In order to identify regulatory genes involved in the development of an apoptosis-resistant phenotype in patients with chemotherapy refractory B-cell chronic lymphocytic leukemia (B-CLL) expression of apoptosis-regulating genes in B-CLL cells was quantified using cDNA arrays and RT-PCR. Data were obtained from and compared between 2 groups of B-CLL patients with either nonprogressive, indolent, previously untreated disease and with leukemic cells sensitive to in vitro fludarabine-induced apoptosis, referred to as sensitive B-CLL (sB-CLL) or with progressive, chemotherapy refractory disease and with leukemic cells resistant to in vitro fludarabine-induced apoptosis, referred to as resistant B-CLL (rB-CLL). By performing a supervised clustering of genes that most strongly discriminated between rB-CLL vs. sB-CLL a small group of genes was identified, where bfl-1 was the strongest discriminating gene (p < 0.05), with higher expression in rB-CLL. A group of apoptosis-regulating genes were modulated during induction of apoptosis by serum deprivation in vitro in a similar manner in all cases studied. However, bfl-1 was preferentially downregulated in sB-CLL as compared to rB-CLL (p < 0.05). We conclude that bfl-1 may be an important regulator of B-CLL apoptosis, which could contribute to disease progression and resistance to chemotherapy, and as such represent a future potential therapeutic target.