Low Molecular Weight Isoforms Research Articles

Abstract PO1-16-04: Targeting PKMYT1 Kinase as a Therapeutic Strategy for Treatment of Triple Negative Breast Cancer with Low Molecular Weight Cyclin E (LMW-E) Expression

Abstract Purpose: Low molecular weight isoforms of cyclin E (LMW-E) have been implicated in various human cancers, including triple negative breast cancer (TNBC), and are associated with a poor prognosis. However, targeted therapies for TNBC based on biomarkers are currently lacking. This study aims to investigate LMW-E as a potential therapeutic target in TNBC and evaluate the efficacy of RP-6306, a selective inhibitor of the Protein Kinase, Membrane Associated Tyrosine/Threonine 1 (PKMYT1), in LMW-E-positive breast tumors. Experimental Design: Immunohistochemical (IHC) analysis was performed on pre-treatment tumor specimens from TNBC patients (n=36) to assess the correlation between LMW-E expression, CDK1 phosphorylation at Threonine 14 (pT14), and pathologic complete response to neoadjuvant chemotherapy. LMW-E inducible human mammary epithelial cells (hMEC) and breast cancer cell lines were used to investigate the regulatory effect of LMW-E on PKMYT1, the kinase responsible for CDK1 phosphorylation at T14, and the response to RP-6306, a first in-class and selective inhibitor of PKMYT1. Patient-derived xenograft (PDX) models and transgenic mouse mammary tumor virus (MMTV) models of TNBC expressing human LMW-E (hLMW-E) were also utilized to assess LMW-E as a biomarker for predicting response to RP-6306. Results. Analysis of TNBC tumor biopsies revealed a significant positive correlation between LMW-E expression and CDK1 pT14, and both biomarkers were associated with a lack of pathological complete response to neoadjuvant chemotherapy. In vitro results using LMW-E inducible hMECs and breast cancer cell lines demonstrated that LMW-E up-regulates PKMYT1 and CDK1 pT14, acting as a PKMYT1 binding protein and enhancing PKMYT1 protein stability. High LMW-E protein levels predicted a favorable response to RP-6306, resulting in the accumulation of sub-G1 and polyploid cells, decreased tolerance to replication stress, increased DNA damage, chromosomal breakage, and apoptosis. In vivo treatment of TNBC PDX models and hLMW-E transgenic tumors with RP-6306 resulted in a significant reduction in tumor volume only in mice harboring high LMW-E tumors, while low cyclin E models showed no response. Immunohistochemical analysis confirmed increased γ-H2AX and decreased CDK1-pT14 and Ki67 levels, indicating the efficacy of RP-6306 in both PDX and transgenic models. Conclusion: This study highlights the regulatory axis from LMW-E to PKMYT1 and its predictive value for pathological complete response in TNBC patients receiving neoadjuvant chemotherapy. The selective PKMYT1 kinase inhibitor RP-6306 consistently induced DNA damage and inhibited tumor growth in in vitro and in vivo pre-clinical breast tumor models. Co-expression of LMW-E and CDK1-pT14 in TNBC can be used to stratify patients whose tumors are likely to respond to RP-6306, emphasizing its therapeutic significance. Citation Format: Mi Li, Amriti Lulla, Cansu Karakas, Spiridon Tsavaschidis, Yan Wang, Tuyen Nguyun, Tuyen Bui, Gary Marshall, Kelly Hunt, Khandan Keyomarsi. Targeting PKMYT1 Kinase as a Therapeutic Strategy for Treatment of Triple Negative Breast Cancer with Low Molecular Weight Cyclin E (LMW-E) Expression [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-16-04.

Abstract 950: Targeting PKMYT1 kinase is an effective treatment strategy in triple negative breast cancers with low molecular weight cyclin E (LMW-E) expression

Abstract Background: Cyclin E is post-translationally modified by neutrophil elastase mediated proteolytic cleavage to generate the low molecular weight isoforms of cyclin E (LMW-E) that are detected in various human cancers. We previously reported that 70% of triple negative breast cancers (TNBC) examined overexpress LMW-E, and these patients have a poor prognosis. Expression of LMW-E promotes genomic instability by causing DNA replication stress. PKMYT1 prevents premature mitotic entry by catalyzing CDK1 phosphorylation at T14, essential for preventing DNA damage and cell death when cyclin E, including LMW-E, is overexpressed. In this study, we tested the hypothesis that LMW-E positive status can be used as a biomarker of response in selecting TNBC patients who are likely to respond to RP-6306, a first in-class and selective inhibitor of PKMYT1 kinase. Results: Assessment of pre-treatment breast biopsies from TNBC patients (n=40) enrolled in a neoadjuvant chemotherapy prospective study for LMW-E and CDK1-pT14 revealed significant positive correlation between these two proteins. Furthermore, positivity of both biomarkers was associated with lack of pathologic complete response (pCR) to neoadjuvant chemotherapy. We next examined the mechanism of response to RP-6306 in vitro and in vivo using TNBC cell lines, patient-derived xenograft (PDX) models and transgenic mouse mammary tumor virus (MMTV) models expressing human LMW-E (hLMW-E). In vitro results using 7 different TNBC cell lines, revealed that high LMW-E levels are significantly predictive of response to RP-6306 (R2=0.78, p= 0.008), while LMW-E knockdown resulted in a 7X increase in IC50 values of RP-6306 (p<0.001). In high LMW-E cells, treatment with RP-6306 resulted in significant (i) downregulation of CDK1-pT14, PKMYT1, WEE1, cyclin B and pRb, (ii) accumulation of sub-G1 and polyploid cell population, (iii) apoptosis, (iv) accumulation of chromosomal breakage, (v) increased DNA damage (increase in γ-H2AX and 53BP1 foci/cell) and lack of DNA repair (downregulation of Rad51), and (vi) premature mitotic entry. Treatment of both breast cancer PDX models and hLMW-E transgenic tumors with RP-6306 revealed that only in animals with high LMW-E tumors, treatment results in significant decrease in tumor volume. However, RP-6306 was ineffective in reducing tumor volume in low cyclin E in vivo models. Immunohistochemical analysis revealed that in vivo efficacy of RP-6306 (in both PDX and transgenic models) was concomitant with increase in γ-H2AX and decrease in CDK1-pT14 and Ki67. Conclusion: Collectively, our results show that overexpression of LMW-E and CDK1-pT14 in TNBC can be used to stratify patients whose tumors are likely to respond to RP-6306. Mechanistically, LMW-E overexpressing TNBC cells activate CDK1 (in vitro and in vivo) to accelerate premature mitotic entry, leading to DNA damage and apoptosis. Citation Format: Amriti Lulla, Tuyen D. Nguyen, Mi Li, Sofia Mastoraki, Yan Wang, Tuyen Bui, Marc Pina, Spiridon Tsavachidis, Gary Marshall, Kelly K. Hunt, Khandan Keyomarsi. Targeting PKMYT1 kinase is an effective treatment strategy in triple negative breast cancers with low molecular weight cyclin E (LMW-E) expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 950.

Regulation of Tau Expression in Superior Cervical Ganglion (SCG) Neurons In Vivo and In Vitro.

The superior cervical ganglion (SCG) is part of the autonomic nervous system providing sympathetic innervation to the head and neck, and has been regularly used to prepare postnatal neuronal cultures for cell biological studies. We found that during development these neurons change tau expression from the low molecular weight (LMW) isoforms to Big tau, with the potential to affect functions associated with tau such as microtubule dynamic and axonal transport. Big tau contains the large 4a exon that transforms tau from LMW isoforms of 45-60 kDa to 110 kDa. We describe tau expression during postnatal development reporting that the transition from LMW tau to Big tau which started at late embryonic stages is completed by about 4-5 weeks postnatally. We confirmed the presence of Big tau in dissociated postnatal SCG neurons making them an ideal system to study the function of Big tau in neurons. We used SCG explants to examine the response of SCG neurons to lesion and found that Big tau expression returned gradually along the regrowing neurites suggesting that it does not drives regeneration, but facilitates the structure/function of mature SCG neurons. The structural/functional roles of Big tau remain unknown, but it is intriguing that neurons that express Big tau appear less vulnerable to tauopathies.

Endothelial TrkA coordinates vascularization and innervation in thermogenic adipose tissue and can be targeted to control metabolism

ObjectiveBrown adipogenesis and thermogenesis in brown and beige adipose tissue (AT) involve vascular remodeling and sympathetic neuronal guidance. Here, we investigated the molecular mechanism coordinating these processes. MethodsWe used mouse models to identify the molecular target of a peptide CPATAERPC homing to the endothelium of brown and beige AT. ResultsWe demonstrate that CPATAERPC mimics nerve growth factor (NGF) and identify a low molecular weight isoform of NGF receptor, TrkA, as the CPATAERPC cell surface target. We show that the expression of truncated endothelial TrkA is selective for brown and subcutaneous AT. Analysis of mice with endothelium-specific TrkA knockout revealed the role of TrkA in neuro-vascular coordination supporting the thermogenic function of brown adipocytes. A hunter-killer peptide D-BAT, composed of CPATAERPC and a pro-apoptotic domain, induced cell death in the endothelium and adipocytes. This resulted in thermogenesis impairment, and predisposed mice to obesity and glucose intolerance. We also tested if this treatment can inhibit the tumor recruitment of lipids mobilized from adipocytes from adjacent AT. Indeed, in a mouse model of breast cancer D-BAT suppressed tumor-associated AT lipolysis, which resulted in reduced fatty acid utilization by cancer cells. ConclusionOur study demonstrates that TrkA signaling in the endothelium supports neuro-vascular coordination enabling beige adipogenesis.

Distinct actin–tropomyosin cofilament populations drive the functional diversification of cytoskeletal myosin motor complexes

SummaryThe effects of N-terminal acetylation of the high molecular weight tropomyosin isoforms Tpm1.6 and Tpm2.1 and the low molecular weight isoforms Tpm1.12, Tpm3.1, and Tpm4.2 on the actin affinity and the thermal stability of actin-tropomyosin cofilaments are described. Furthermore, we show how the exchange of cytoskeletal tropomyosin isoforms and their N-terminal acetylation affects the kinetic and chemomechanical properties of cytoskeletal actin-tropomyosin-myosin complexes. Our results reveal the extent to which the different actin-tropomyosin-myosin complexes differ in their kinetic and functional properties. The maximum sliding velocity of the actin filament as well as the optimal motor density for continuous unidirectional movement, parameters that were previously considered to be unique and invariant properties of each myosin isoform, are shown to be influenced by the exchange of the tropomyosin isoform and the N-terminal acetylation of tropomyosin.

Adiponectin Inhibits the Production of TNF-α, IL-6 and Chemokines by Human Lung Macrophages.

Background: Obesity is associated with an elevated risk of severe respiratory infections and inflammatory lung diseases. The objectives were to investigate 1) the production of adiponectin by human lung explants, 2) the expression of the adiponectin receptors AdipoR1 and AdipoR2 by human lung macrophages (LMs), and 3) the impact of recombinant human adiponectin and a small-molecule APN receptor agonist (AdipoRon) on LMs activation.Material and methods: Human parenchyma explants and LMs were isolated from patients operated for carcinoma. The LMs were cultured with recombinant adiponectin or AdipoRon and stimulated with lipopolysaccharide (10 ng ml−1), poly (I:C) (10 µg ml−1) or interleukin (IL)-4 (10 ng ml−1) for 24 h. Cytokines or adiponectin, released by explants or LMs, were measured using ELISAs. The mRNA levels of AdipoR1 and AdipoR2 were determined using real-time quantitative PCR. AdipoRs expression was also assessed with confocal microscopy.Results: Adiponectin was released by lung explants at a level negatively correlated with the donor’s body mass index. AdipoR1 and AdipoR2 were both expressed in LMs. Adiponectin (3–30 µg ml−1) and AdipoRon (25–50 μM) markedly inhibited the LPS- and poly (I:C)-induced release of Tumor Necrosis Factor-α, IL-6 and chemokines (CCL3, CCL4, CCL5, CXCL1, CXCL8, CXCL10) and the IL-4-induced release of chemokines (CCL13, CCL17, CCL22) in a concentration-dependent manner. Recombinant adiponectin produced in mammalian cells (lacking low molecular weight isoforms) had no effects on LMs.Conclusion and implications: The low-molecular-weight isoforms of adiponectin and AdipoRon have an anti-inflammatory activity in the lung environment. Targeting adiponectin receptors may constitute a new means of controlling airways inflammation.

Low-molecular-weight fibroblast growth factor-2 — a viable prognostic factor for gastric gastrointestinal stromal tumors

Aim. To examine the expression of fibroblast growth factor-2 and its isoforms in gastrointestinal stromal tumors and assess the prognostic value of this marker.
 Methods. The study included 44 patients with gastric gastrointestinal stromal tumors of the stomach who were prescribed surgical or combined treatment with the targeted drug imatinib (imatinib mesylate). Immunohistochemistry (IHC)-staining and immunoblotting with monoclonal antibodies were used to assess the expression of FGF-2. Statistical analysis for differences in clinical and morphological parameters was performed by using Students, MannWhitneyWilcoxon and Fishers tests. Differences were considered significant at p 0.05.
 Results. Fibroblast growth factor-2 expression was assessed in tumor tissues in 39 out of 44 analyzed patients. The frequency of fibroblast growth factor-2 expression in the observed patients was 84.6% (33/39). The moderate and strong fibroblast growth factor-2 expression was detected in 21 (53.8%) patients with gastric gastrointestinal stromal tumors. High expression of low-molecular weight (18 kDa) fibroblast growth factor-2 isoform was found in all tumor samples from patients with high-risk gastrointestinal stromal tumor (prognostic group 6) (p=0.039), which indicated the active secretion of this ligand by its signalling pathway in the cancer cells. Patients with high levels of low‐molecular‐weight fibroblast growth factor-2 showed a higher level of Ki-67 proliferative activity (р=0.013) and tumor size (р=0.0017). Patients with increased expression of the low molecular weight isoform of fibroblast growth factor-2 in gastric gastrointestinal stromal tumor had a higher risk of recurrence, as well as larger tumor size and proliferative activity compared with patients without expression of this isoform. The level of fibroblast growth factor-2 expression in tumor samples, determined by immunohistochemistry-staining, increases after initiation of imatinib to based therapy, which may indicate the formation of resistance to this targeted drug and the progression of the disease.
 Conclusion. The results of the study suggest that FGF-2 might be an independent prognostic marker of gastric gastrointestinal stromal tumor and a viable therapeutic target.

Elimination of endogenous high molecular weight FGF2 prevents pressure-overload-induced systolic dysfunction, linked to increased FGFR1 activity and NR1D1 expression.

Fibroblast growth factor 2 (FGF2), produced as high (Hi-) and low (Lo-) molecular weight isoforms, is implicated in cardiac response to injury. The role of endogenous FGF2 isoforms during chronic stress is not well defined. We investigated the effects of endogenous Hi-FGF2 in a mouse model of simulated pressure-overload stress achieved by transverse aortic constriction (TAC) surgery. Hi-FGF2 knockout mice, expressing only Lo-FGF2, FGF2(Lo), and wild-type mice, FGF2(WT), expressing both Hi-FGF2 and Lo-FGF2, were used. By echocardiography, a decline in systolic function was observed in FGF2(WT) but not FGF2(Lo) mice compared to corresponding sham-operated animals at 4-8weeks post-TAC surgery. TAC surgery increased markers of myocardial stress/damage including B-type natriuretic peptide (BNP) and the pro-cell death protein BCL2/adenovirus E1B 19kDa protein-interacting protein-3 (Bnip3) in FGF2(WT) but not FGF2(Lo) mice. In FGF2(Lo) mice, cardiac levels of activated FGF receptor 1 (FGFR1), and downstream signals, including phosphorylated mTOR and p70S6 kinase, were elevated post-TAC. Finally, NR1D1 (nuclear receptor subfamily 1 group D member 1), implicated in cardioprotection from pressure-overload stress, was downregulated or upregulated in the presence or absence, respectively, of Hi-FGF2 expression, post-TAC surgery. In wild-type cardiomyocyte cultures, endothelin-1 (added to simulate pressure-overload signals) caused NR1D1 downregulation and BNP upregulation, similar to the effect of TAC surgery on the FGF2(WT) mice. The NR1D1 agonist SR9009 prevented BNP upregulation, simulating post-TAC findings in FGF2(Lo) mice. We propose that elimination of Hi-FGF2 is cardioprotective during pressure-overload by increasing FGFR1-associated signaling and NR1D1 expression.

Review on Adiponectin: A Benevolent Adipokine

Adiponectin is a most abundant secretory protein produced by adipocytes of white adipose tissue. Adiponectin circulates in blood as three different (high-molecular, middle-molecular, and low-molecular weight) isoforms, gives its effects through AdipoR1 and AdipoR2 receptor. Primary data suggesting that adiponectin has insulin-sensitizing, anti-atherogenic, and anti-inflammatory effects. High serum level of adiponectin is positively associated with inflammation severity and pathological progression in chronic kidney disease, liver disease and inflammatory bowel disease. It has emerged as a valuable biomarker for insulin sensitivity, cardiovascular risk and inflammation. Adiponectin is gaining attention for its therapeutic role in Alzheimer’s disease. Adiponectin appears to play a crucial role not only in glucose and lipid metabolism but also the development and progression of different cancers. Adiponectin also produced locally in the retinas participate in defense of various eye diseases. This review summarizes the role of adiponectin as benevolent adipokine in different disorders.

Healing and Angiogenic Properties of Collagen/Chitosan Scaffolds Enriched with Hyperstable FGF2-STAB® Protein: In Vitro, Ex Ovo and In Vivo Comprehensive Evaluation.

Wound healing is a process regulated by a complex interaction of multiple growth factors including fibroblast growth factor 2 (FGF2). Although FGF2 appears in several tissue engineered studies, its applications are limited due to its low stability both in vitro and in vivo. Here, this shortcoming is overcome by a unique nine-point mutant of the low molecular weight isoform FGF2 retaining full biological activity even after twenty days at 37 °C. Crosslinked freeze-dried 3D porous collagen/chitosan scaffolds enriched with this hyper stable recombinant human protein named FGF2-STAB® were tested for in vitro biocompatibility and cytotoxicity using murine 3T3-A31 fibroblasts, for angiogenic potential using an ex ovo chick chorioallantoic membrane assay and for wound healing in vivo with 3-month old white New Zealand rabbits. Metabolic activity assays indicated the positive effect of FGF2-STAB® already at very low concentrations (0.01 µg/mL). The angiogenic properties examined ex ovo showed enhanced vascularization of the tested scaffolds. Histological evaluation and gene expression analysis by RT-qPCR proved newly formed granulation tissue at the place of a previous skin defect without significant inflammation infiltration in vivo. This work highlights the safety and biocompatibility of newly developed crosslinked collagen/chitosan scaffolds involving FGF2-STAB® protein. Moreover, these sponges could be used as scaffolds for growing cells for dermis replacement, where neovascularization is a crucial parameter for successful skin regeneration.

Depletion of androgen receptor low molecular weight isoform reduces bladder tumor cell viability and induces apoptosis.

Bladder cancer (BlCa) exhibits a gender disparity where men are three times more likely to develop the malignancy than women suggesting a role for the androgen receptor (AR). Here we report that BlCa cells express low molecular weight (LMW) AR isoforms that are missing the ligand binding domain (LBD). Isoform expression was detected in most BlCa cells, while a few express the full-length AR. Immunofluorescence studies detect AR in the nucleus and cytoplasm, and localization is cell dependent. Cells with nuclear AR expression exhibit reduced viability and increased apoptosis on total AR depletion. A novel AR-LMW variant, AR-v19, that is missing the LBD and contains 15 additional amino acids encoded by intron 3 sequences was detected in most BlCa malignancies. AR-v19 localizes to the nucleus and can transactivate AR-dependent transcription in a dose dependent manner. AR-v19 depletion impairs cell viability and promotes apoptosis in cells that express this variant. Thus, AR splice variant expression is common in BlCa and instrumental in ensuring cell survival. This suggests that targeting AR or AR downstream effectors may be a therapeutic strategy for the treatment of this malignancy.

Alternatively Spliced Form of Angiopoietin-2 as a New Vascular Rheostat.

Angiopoietin (ANPGT)-TIE signaling serves as a critical regulator of vessel maturation controlling vascular quiescence, maintenance, and homeostasis (primarily through ANGPT1-TIE2 signaling), as well as enabling vascular plasticity and responsiveness to exogenous cytokines (primarily through antagonistically acting ANGPT2). An alternatively spliced form of ANGPT2 (ANGPT2443) was first reported 20 years ago. Yet, little is known to this day about its biological functions. In this issue of Cancer Research, Kapiainen and colleagues report an elegant series of experiments adding to the complexity and contextuality of ANGPT-TIE signaling. The authors studied the function of ANGPT2443 in cellular experiments as well as in a genetic model in vivo, revealing that it is proteolytically cleaved into a lower molecular weight isoform (termed ANGPT2DAP) that lacks the superclustering domain necessary for multimer formation. When compared with full-length ANGPT2, ANGPT2443 and ANGPT2DAP showed lower binding affinity to α5β1 integrin, but were more potent inhibitors of ANGPT1-TIE2 signaling. Functionally, ANGPT2443 impaired vessel enlargement and vein morphogenesis during postnatal retinal angiogenesis. Tumor experiments in Angpt2443-expressing mice showed enhanced destabilization of the lung vasculature, with varying effects on metastasis. Taken together, the study provides important insight into the significance of ANGPT2 alternative splicing and identifies ANGPT2443 and ANGPT2DAP as a biological rheostat of ANGPT1-TIE2 signaling. Future work will need to characterize the relative ratios and functional contributions of the ANGPT2 variants in different pathophysiologic settings.See related article by Kapiainen et al., p. 129.

What Can We Learn from FGF-2 Isoform-Specific Mouse Mutants? Differential Insights into FGF-2 Physiology In Vivo.

Fibroblast growth factor 2 (FGF-2), ubiquitously expressed in humans and mice, is functionally involved in cell growth, migration and maturation in vitro and in vivo. Based on the same mRNA, an 18-kilo Dalton (kDa) FGF-2 isoform named FGF-2 low molecular weight (FGF-2LMW) isoform is translated in humans and rodents. Additionally, two larger isoforms weighing 21 and 22 kDa also exist, summarized as the FGF-2 high molecular weight (FGF-2HMW) isoform. Meanwhile, the human FGF-2HMW comprises a 22, 23, 24 and 34 kDa protein. Independent studies verified a specific intracellular localization, mode of action and tissue-specific spatiotemporal expression of the FGF-2 isoforms, increasing the complexity of their physiological and pathophysiological roles. In order to analyze their spectrum of effects, FGF-2LMW knock out (ko) and FGF-2HMWko mice have been generated, as well as mice specifically overexpressing either FGF-2LMW or FGF-2HMW. So far, the development and functionality of the cardiovascular system, bone formation and regeneration as well as their impact on the central nervous system including disease models of neurodegeneration, have been examined. This review provides a summary of the studies characterizing the in vivo effects modulated by the FGF-2 isoforms and, thus, offers a comprehensive overview of its actions in the aforementioned organ systems.

The effects of blanching on composition and modification of proteins in navy beans (Phaseolus vulgaris)

Blanching is an important process in the preparation of navy beans (Phaseolus vulgaris L.) for canning. We here explore the effect of blanching which can profoundly affect protein composition and introduce protein-primary-level modifications. Amino acid analysis showed significantly decreased protein abundance (58.5%) in blanched beans compared to raw beans. Proteomic analyses revealed a decrease in high molecular weight isoforms of the major storage globulin proteins phaseolin (mean fold-change −3.7) and legumin (mean fold-change −2.5) and concomitant increase in their low molecular weight isoforms (mean fold-change 6.4 and 8.3, respectively). Blanched beans also had decreased abundance of lipoxygenase (mean fold-change −13.1), an enzyme responsible for product spoilage during storage. Increased lysinoalanine (up to 47%) and highly modified protein fragments were found in the processing waters, indicating heat- induced modifications. Correlating these molecular level changes thus provides a basis for evaluating how processing parameters can be modified to increase protein food quality.

Mini-Review: The Contribution of Adipokines to Joint Inflammation in Inflammatory Rheumatic Diseases.

Inflammatory rheumatic diseases (IRD) are complex disorders characterized by chronic inflammation of the joints and related skeletal structures. The most common forms of IRD are rheumatoid arthritis (RA) and spondyloarthritis (SpA), including axial SpA (axSpA) and psoriatic arthritis (PsA). Obesity is a frequent comorbidity in RA and PsA, and to a lesser extend in axial SpA. The association between obesity and IRD may be explained by the release from fat tissue of several bioactive proteins, namely adipokines. Adipokines are involved in the regulation of various processes such as lipid or glucose metabolism, but also inflammation. Adipokines are interrelated with the immune system, with both innate and adaptive immune cell connections. Several adipokines with pro-inflammatory effects have been identified such as leptin, visfatin or resistin. Conversely, adiponectin and more specifically its low molecular weight isoform, is considered to have antiinflammatory properties. In this review, we discuss the contribution of adipokines to the joint inflammation of IRD, the relation they have with immune pathways of these diseases, their links with the structural impact on peripheral joints and/or axial skeleton, and also the influence they may have on the cardiometabolic risk of IRD.

Phosphoproteomic analysis identifies phospho-Threonine-17 site of phospholamban important in low molecular weight isoform of fibroblast growth factor 2-induced protection against post-ischemic cardiac dysfunction

RationaleAmong its many biological roles, fibroblast growth factor 2 (FGF2) protects the heart from dysfunction and damage associated with an ischemic attack. Our laboratory demonstrated that its protection against myocardial dysfunction occurs by the low molecular weight (LMW) isoform of FGF2, while the high molecular weight (HMW) isoforms are associated with a worsening in post-ischemic recovery of cardiac function. LMW FGF2-mediated cardioprotection is facilitated by activation of multiple kinases, including PKCalpha, PKCepsilon, and ERK, and inhibition of p38 and JNK. ObjectiveYet, the substrates of those kinases associated with LMW FGF2-induced cardioprotection against myocardial dysfunction remain to be elucidated. Methods and resultsTo identify substrates in LMW FGF2 improvement of post-ischemic cardiac function, mouse hearts expressing only LMW FGF2 were subjected to ischemia-reperfusion (I/R) injury and analyzed by a mass spectrometry (MS)-based quantitative phosphoproteomic strategy. MS analysis identified 50 phosphorylation sites from 7 sarcoendoplasmic reticulum (SR) proteins that were significantly altered in I/R-treated hearts only expressing LMW FGF2 compared to those hearts lacking FGF2. One of those phosphorylated SR proteins identified was phospholamban (PLB), which exhibited rapid, increased phosphorylation at Threonine-17 (Thr17) after I/R in hearts expressing only LMW FGF2; this was further validated using Selected Reaction Monitoring-based MS workflow. To demonstrate a mechanistic role of phospho-Thr17 PLB in LMW FGF2-mediated cardioprotection, hearts only expressing LMW FGF2 and those expressing only LMW FGF2 with a mutant PLB lacking phosphorylatable Thr17 (Thr17Ala PLB) were subjected to I/R. Hearts only expressing LMW FGF2 showed significantly improved recovery of cardiac function following I/R (p < 0.05), and this functional improvement was significantly abrogated in hearts expressing LMW FGF2 and Thr17Ala PLB (p < 0.05). ConclusionThe findings indicate that LMW FGF2 modulates intracellular calcium handling/cycling via regulatory changes in SR proteins essential for recovery from I/R injury, and thereby protects the heart from post-ischemic cardiac dysfunction.

Changes in gene expression caused by genetic elimination of high molecular weight FGF2 are associated with prevention of stress‐induced cardiac systolic dysfunction

Fibroblast growth factor 2 (FGF2) is produced as high (&gt;20 kDa, Hi‐) and low molecular weight (18 kDa, Lo) isoforms in the heart. While administered Lo‐FGF2 has been established as a cardioprotective agent in multiple models of cardiac injury, there is limited information about the role of Hi‐FGF2 in the heart. To investigate the effect of endogenous Hi‐FGF2 we compared cardiac transcriptome (35240 targets, GeneChip™ Mouse Gene 2.0 ST Array (Affymetrix, 902119)) and systolic function between Hi‐FGF2 knock‐out mice, FGF2(Lo), expressing only Lo‐FGF2, and wild type mice, FGF2(WT), expressing both Hi‐FGF2 and Lo‐FGF2, under ‘normal’ (sham‐operated) and “stress” (pressure overload) conditions. Transaortic constriction surgery (TAC) was used to induce pressure overload. Echocardiography was done at baseline and at 4–8 weeks post‐surgery. Microarray analysis showed that under normal conditions, the absence of Hi‐FGF2 promoted changes in relative levels of 118 transcripts, including those associated with circadian rhythm regulation and heat shock protein (HSP70)‐associated apoptosis regulation. Comparative analysis of TAC surgery‐induced gene expression changes showed that 275 transcripts were differentially affected by the presence or absence of Hi‐FGF2 expression, most prominently the orphan nuclear receptor NR1D1 which is linked to regulation of circadian rhythm and metabolism. Under non‐stress conditions, systolic function was unaffected by endogenous Hi‐FGF2 expression. Pressure overload stress caused a decline in systolic function in the presence of endogenous Hi‐FGF2, at 4–8 weeks post‐TAC surgery, accompanied by increases in markers of myocardial stress/damage including B‐type natriuretic peptide (BNP) and the pro‐cell death protein BCL2/adenovirus E1B 19 kDa protein‐interacting protein‐3, Bnip3. In the absence of endogenous Hi‐FGF2, mice were protected from stress‐induced loss of systolic function and increases in BNP and Bnip3. TAC surgery induced Hi‐FGF2‐independent increases in: cardiac mass (heart weight/tibia length); cardiac fibrosis; transcripts linked to exracellular matrix remodeling. Increased cardiomyocyte size, however, was only observed in FGF2(WT) but not FGF2(Lo) hearts post‐TAC. It is suggested that elimination of endogenous Hi‐FGF2 elicits cardioprotection by increasing cardiac HSP70 (pre‐TAC surgery); and NR1D1, after TAC surgery.Support or Funding InformationFunding (EK, PAC, DJ) was provided by the Canadian Institutes for Health Research (FRN‐74733) and the Molson Women’s Heart Health Foundation (EK). MPC was supported by a CIHR Open Operating Grant (MOP136862). NK and RSN were the recipients of a Bank of Montreal studentship award via the St. Boniface Hospital Albrechtsen Research Centre and University of Manitoba funding to PAC.

Multifaceted Physiological Roles of Adiponectin in Inflammation and Diseases.

Adiponectin is the richest adipokine in human plasma, and it is mainly secreted from white adipose tissue. Adiponectin circulates in blood as high-molecular, middle-molecular, and low-molecular weight isoforms. Numerous studies have demonstrated its insulin-sensitizing, anti-atherogenic, and anti-inflammatory effects. Additionally, decreased serum levels of adiponectin is associated with chronic inflammation of metabolic disorders including Type 2 diabetes, obesity, and atherosclerosis. However, recent studies showed that adiponectin could have pro-inflammatory roles in patients with autoimmune diseases. In particular, its high serum level was positively associated with inflammation severity and pathological progression in rheumatoid arthritis, chronic kidney disease, and inflammatory bowel disease. Thus, adiponectin seems to have both pro-inflammatory and anti-inflammatory effects. This indirectly indicates that adiponectin has different physiological roles according to an isoform and effector tissue. Knowledge on the specific functions of isoforms would help develop potential anti-inflammatory therapeutics to target specific adiponectin isoforms against metabolic disorders and autoimmune diseases. This review summarizes the current roles of adiponectin in metabolic disorders and autoimmune diseases.

Abstract 838: High Molecular Weight FGF2 Contributes to Pressure Overload Induced Systolic Dysfunction by a Mechanism Associated With Modulation of the NR1D1 Orphan Nuclear Receptor Expression

Fibroblast growth factor 2 (FGF2) is implicated in normal cardiac development as well as cardiac pathophysiology; however, FGF2 exist as multiple high and low molecular weight isoforms. While endogenous low molecular weight FGF2 (Lo-FGF2) is cardioprotective during chronic stress, the more prevalent endogenous high molecular weight FGF2 (Hi-FGF2) is proposed to promote maladaptive cardiac remodeling. We have investigated the hypothesis that genetic elimination of Hi-FGF attenuates cardiac dysfunction in mice that have been subjected to pressure overload by transverse aortic constriction (TAC). Two groups of male C57BL/6mice were compared: (1) Wild type (WT) mice, expressing Hi- and Lo-FGF2 (FGF[WT] mice); and (2) Hi-FGF2 knock-out mice, expressing only Lo-FGF2 (FGF[Lo] mice). Echocardiographic assessment of heart function and dimensions was done at baseline and then 4 and 8 weeks after TAC or sham surgery. FGF[WT] mice displayed a decline in systolic function compared to their corresponding sham animals at 4- and 8-weeks post-TAC, which was absent in the FGF[Lo] mice. Relative levels of B-type natriuretic peptide, a marker of cardiac pathology severity, were elevated in FGF[WT] but not FGF[Lo] mice compared to shams. Increased accumulation of the pro-cell death protein BCL2/adenovirus E1B 19 kDa protein-interacting protein-3 was more pronounced in the FGF(WT) compared to FGF(Lo) mice, post TAC. Microarray analysis of the whole transcriptome of hearts in FGF2[WT] and FGF2[Lo] mice indicated the pathway linked to circadian rhythm as a candidate for the most significant differentially regulated. Specifically, upregulation of the circadian rhythm master regulator, Nuclear Receptor Subfamily 1 Group D Member 1 (NR1D1), was validated by qPCR and protein immunoblotting in FGF[Lo] mice versus downregulation of NR1D1in FGF[WT] mice post-TAC, when compared to their sham operated littermates. Taken together these studies suggest that expression of Hi-FGF2 contributes to cardiac systolic dysfunction in left ventricular pressure overloaded WT mice by downregulation of Nr1D1, post-TAC.

Astrocytes Modify Migration of PBMCs Induced by β-Amyloid in a Blood-Brain Barrier in vitro Model.

BackgroundThe brain is protected by the blood-brain barrier (BBB), constituted by endothelial cells supported by pericytes and astrocytes. In Alzheimer’s disease a dysregulation of the BBB occurs since the early phases of the disease leading to an increased access of solutes and immune cells that can participate to the central inflammatory response. Here we investigated whether astrocytes may influence endothelial-leukocytes interaction in the presence of amyloid-β (Aβ).MethodsWe used an in vitro BBB model, where endothelial cells, cultured alone or with astrocytes were exposed for 5 h to Aβ, both under resting or inflammatory conditions (TNFα and IFNγ), to evaluate endothelial barrier properties, as well as transendothelial migration of peripheral blood mononuclear cells (PBMCs).ResultsIn the co-culture model, barrier permeability to solutes was increased by all treatments, but migration was only observed in inflammatory conditions and was prevented by Aβ treatment. On the contrary, in endothelial monocultures, Aβ induced leukocytes migration under resting conditions and did not modify that induced by inflammatory cytokines. In endothelial astrocyte co-cultures, a low molecular weight (MW) isoform of the adhesion molecule ICAM-1, important to allow interaction with PBMCs, was increased after 5 h exposure to inflammatory cytokines, an effect that was prevented by Aβ. This modulation by Aβ was not observed in endothelial monocultures. In addition, endothelial expression of β-1,4-N-acetylglucosaminyltransferase III (Gnt-III), responsible for the formation of the low MW ICAM-1 isoform, was enhanced in inflammatory conditions, but negatively modulated by Aβ only in the co-culture model. miR-200b, increased in astrocytes following Aβ treatment and may represent one of the factors involved in the control of Gnt-III expression.ConclusionThese data point out that, at least in the early phases of Aβ exposure, astrocytes play a role in the modulation of leukocytes migration through the endothelial layer.