CDK1 inhibition reduces osteogenesis in endothelial cells in vascular calcification

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Abstract

Vascular calcification is a severe complication of cardiovascular diseases. Previous studies demonstrated that endothelial lineage cells transitioned into osteoblast-like cells and contributed to vascular calcification. Here, we found that inhibition of cyclin-dependent kinase (CDK) prevented endothelial lineage cells from transitioning to osteoblast-like cells and reduced vascular calcification. We identified a robust induction of CDK1 in endothelial cells (ECs) in calcified arteries and showed that endothelial-specific gene deletion of CDK1 decreased the calcification. We found that limiting CDK1 induced E-twenty-six specific sequence variant 2 (ETV2), which was responsible for blocking endothelial lineage cells from undergoing osteoblast differentiation. We also found that inhibition of CDK1 reduced vascular calcification in a diabetic mouse model. Together, the results highlight the importance of CDK1 suppression and suggest CDK1 inhibition as a potential option for treating vascular calcification.

Authors

Yan Zhao, Yang Yang, Xiuju Wu, Li Zhang, Xinjiang Cai, Jaden Ji, Sydney Chen, Abigail Vera, Kristina I. Boström, Yucheng Yao

Inhibiting centrosome clustering reduces cystogenesis and improves kidney function in Autosomal Dominant Polycystic Kidney Disease

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Abstract

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a monogenic disorder accounting for approximately 5% of patients with renal failure. Yet, therapeutics for the treatment of ADPKD remain limited. ADPKD tissues display abnormalities in the biogenesis of the centrosome, a defect that can cause genome instability, aberrant ciliary signaling, and secretion of pro-inflammatory factors. Cystic cells form excess centrosomes via a process termed centrosome amplification (CA), which causes abnormal multipolar spindle configurations, mitotic catastrophe, and reduced cell viability. However, cells with CA can suppress multipolarity via “centrosome clustering,” a key mechanism by which cells circumvent apoptosis. Here, we demonstrate that inhibiting centrosome clustering can counteract the proliferation of renal cystic cells with high incidences of CA. Using ADPKD human cells and mouse models, we show that preventing centrosome clustering with two inhibitors, CCB02 and PJ34, blocks cyst initiation and growth in vitro and in vivo. Inhibiting centrosome clustering activates a p53-mediated surveillance mechanism leading to apoptosis, reduced cyst expansion, interstitial fibrosis, and improved kidney function. Transcriptional analysis of kidneys from treated mice identified pro-inflammatory signaling pathways implicated in CA-mediated cystogenesis and fibrosis. Our results demonstrate that centrosome clustering is a cyst-selective target for the improvement of renal morphology and function in ADPKD.

Authors

Tao Cheng, Aruljothi Mariappan, Ewa Langner, Kyuhwan Shim, Jay Gopalakrishnan, Moe R. Mahjoub

A T cell-based SARS-CoV-2 spike protein vaccine provides protection without antibodies

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Abstract

SARS-CoV-2 spike-based vaccines are used to control the COVID-19 pandemic. However, emerging variants became resistant to antibody neutralization and further mutations may lead to full resistance. We tested whether T cells alone could provide protection without antibodies. We designed a T cell-based vaccine in which SARS-CoV-2 spike sequences were rearranged and attached to ubiquitin. Immunization of mice with the vaccine induced no specific antibodies but strong specific T cell responses. We challenged mice with SARS-CoV-2 wild-type strain or an Omicron variant after the immunization and monitored survival or viral titers in the lungs. The mice were significantly protected against death and weight loss caused by SARS-CoV-2 wild-type strain, and the viral titers in the lungs of mice challenged with SARS-CoV-2 wild-type or the Omicron variant were significantly reduced. Importantly, depletion of CD4+ or CD8+ T cells led to significant loss of the protection. Our analyses of spike protein sequences of the variants indicated that fewer than 1/3 presented by dominant HLA alleles were mutated and that most of the mutated epitopes were in subunit 1 region. As subunit 2 region is conservative, the vaccines targeting spike protein are expected to protect against future variants due to the T cell responses.

Authors

Juan Shi, Jian Zheng, Xiujuan Zhang, Wanbo Tai, Ryan Compas, Jack C. Deno, Natalie Jachym, Abhishek K. Verma, Gang Wang, Xiaoqing Guan, Abby E. Odle, Yushun Wan, Fang Li, Stanley Perlman, Liang Qiao, Lanying Du

AXL inhibition suppresses early allograft monocyte-to-macrophage differentiation and prolongs allograft survival

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Abstract

Innate immune cells are important in the initiation and potentiation of alloimmunity in transplantation. Immediately upon organ anastomosis and reperfusion, recipient monocytes enter the graft from circulation and differentiate to inflammatory macrophages to promote allograft inflammation. However, factors that drive their differentiation to inflammatory macrophages are not understood. Here, we showed that the receptor tyrosine kinase AXL was a key driver of early intragraft differentiation of recipient infiltrating monocytes to inflammatory macrophages in the presence of allogeneic stimulation and cell-cell contact. In this context, the differentiated inflammatory macrophages were capable of efficient alloantigen presentation and allo-stimulation of T cells of the indirect pathway. Consequently, early and transient AXL inhibition with the pharmacological inhibitor bemcentinib resulted in a profound reduction of initial allograft inflammation and a significant prolongation of allograft survival in a murine heart transplant model. Our results support further investigation of AXL inhibition as part of an induction regimen for transplantation.

Authors

Collin Z. Jordan, Matthew Tunbridge, Irma Husain, Hiroki Kitai, Miriam E. Dilts, Olivia K. Fay, Koki Abe, Catherine Xiang, Jean Kwun, Tomokazu Souma, Edward B. Thorp, Xunrong Luo

Myeloid Zfhx3 deficiency protects against hypercapnia-induced suppression of host defense against influenza A virus

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Abstract

Hypercapnia, elevation of the partial pressure of CO2 in blood and tissues, is a risk factor for mortality in patients with severe acute and chronic lung diseases. We previously showed that hypercapnia inhibits multiple macrophage and neutrophil antimicrobial functions, and that elevated CO2 increases the mortality of bacterial and viral pneumonia in mice. Here, we show that normoxic hypercapnia downregulates innate immune and antiviral gene programs in alveolar macrophages (AMØs). We also show that zinc finger homeobox 3 (Zfhx3), a mammalian ortholog of zfh2, which mediates hypercapnic immune suppression in Drosophila, is expressed in mouse and human macrophages. Deletion of Zfhx3 in the myeloid lineage blocked the suppressive effect of hypercapnia on immune gene expression in AMØs and decreased viral replication, inflammatory lung injury and mortality in hypercapnic mice infected with influenza A virus. Our results establish Zfhx3 as the first known mammalian mediator of CO2 effects on immune gene expression and lay the basis for future studies to identify therapeutic targets to interrupt hypercapnic immunosuppression in patients with advanced lung disease.

Authors

S. Marina Casalino-Matsuda, Fei Chen, Francisco J. Gonzalez-Gonzalez, Hiroaki Matsuda, Aisha Nair, Hiam Abdala-Valencia, G.R. Scott Budinger, Jin-Tang Dong, Greg J. Beitel, Peter H.S. Sporn

Catalytic isoforms of AMP-activated protein kinase differentially regulate IMPDH activity and photoreceptor neuron function

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Abstract

AMP-activated protein kinase (AMPK) plays a crucial role in maintaining ATP homeostasis in photoreceptor neurons. AMPK is a heterotrimeric protein consisting of alpha, beta, and gamma subunits. The independent functions of the two isoforms of the catalytic alpha subunit, PRKAA1 and PRKAA2, are uncharacterized in specialized neurons such as photoreceptors. Here we demonstrate in mice that rod photoreceptors lacking PRKAA2, but not PRKAA1, show altered levels of cGMP, GTP, and ATP, suggesting isoform-specific regulation of photoreceptor metabolism. Furthermore, PRKAA2 deficient mice display visual functional deficits on electroretinography and photoreceptor outer segment structural abnormalities on transmission electron microscopy consistent with neuronal dysfunction, but not neurodegeneration. Phosphoproteomics identified inosine monophosphate dehydrogenase (IMPDH) as a molecular driver of PRKAA2-specific photoreceptor dysfunction, and inhibition of IMPDH improved visual function in Prkaa2 rod photoreceptor knockout mice. These findings highlight a novel, therapeutically targetable PRKAA2 isoform-specific function of AMPK in regulating photoreceptor metabolism and function through a previously uncharacterized mechanism affecting IMPDH activity.

Authors

Tae Jun Lee, Yo Sasaki, Philip A. Ruzycki, Norimitsu Ban, Joseph B. Lin, Hung-Ting Wu, Andrea Santeford, Rajendra S. Apte

Circular RNA Cdr1as inhibits proliferation and delays injury-induced regeneration of the intestinal epithelium

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Abstract

Circular RNAs (circRNAs) are highly expressed in the mammalian intestinal epithelium, but their functions remain largely unknown. Here we identified the circRNA Cdr1as as a repressor of intestinal epithelial regeneration and defense. Cdr1as levels increase in mouse intestinal mucosa after colitis and septic stress, as well as in human intestinal mucosa from patients with inflammatory bowel diseases and sepsis. Ablation of the Cdr1as locus from the mouse genome enhances renewal of the intestinal mucosa, promotes injury-induced epithelial regeneration, and protects the mucosa against colitis. We found approximately 40 microRNAs, including microRNA miR-195, differentially express between intestinal mucosa of Cdr1as knockout (–/–) versus littermate mice. Increasing the levels of Cdr1as inhibits intestinal epithelial repair after wounding in cultured cells and represses growth of intestinal organoids cultured ex vivo, but this inhibition is abolished by miR-195 silencing. The reduction in miR-195 levels in the Cdr1as–/– intestinal epithelium is the result of reduced stability and processing of the precursor miR-195. These findings indicate that Cdr1as reduces proliferation and repair of the intestinal epithelium at least in part via interaction with miR-195 and highlight a role for induced Cdr1as in the pathogenesis of unhealed wounds and disrupted renewal of the intestinal mucosa.

Authors

Hee Kyoung Chung, Lan Xiao, Naomi Han, Jason Chen, Vivian Yao, Cassandra M. Cairns, Benjamin Raufman, Jaladanki N. Rao, Douglas J. Turner, Rosemary Kozar, Myriam Gorospe, Jian-Ying Wang

C3aR-initiated signaling is a critical mechanism of podocyte injury in membranous nephropathy

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Abstract

The deposition of anti-podocyte auto-antibodies in the glomerular subepithelial space induces primary membranous nephropathy (MN), the leading cause of nephrotic syndrome worldwide. Taking advantage of the glomerulus-on-a-chip system, we modeled human primary MN induced by anti-PLA2R antibodies. Here we show that exposure of primary human podocytes expressing PLA2R to MN serum results in IgG deposition and complement activation on their surface, leading to loss of the chip permselectivity to albumin. C3a receptor (C3aR) antagonists as well as C3AR gene silencing in podocytes reduced oxidative stress induced by MN serum and prevented albumin leakage. In contrast, inhibition of the formation of the membrane-attack-complex (MAC), previously thought to play a major role in MN pathogenesis, did not affect permselectivity to albumin. In addition, treatment with a C3aR antagonist effectively prevented proteinuria in a mouse model of MN, substantiating the chip findings. In conclusion, using a combination of pathophysiologically relevant in vitro and in vivo models, we established that C3a/C3aR signaling plays a critical role in complement-mediated MN pathogenesis, indicating an alternative therapeutic target for MN.

Authors

Qi Zhang, Sofia Bin, Kelly L. Budge, Astgik Petrosyan, Valentina Villani, Paola Aguiari, Coralien H. Vink, Jack Wetzels, Hasmik Soloyan, Gaetano La Manna, Manuel Alfredo Podestà, Paolo Molinari, Sargis Sedrakyan, Kevin V. Lemley, Roger E. De Filippo, Laura Perin, Paolo Cravedi, Stefano Da Sacco

CD3-downregulation identifies T-helper-cells with superior functionality and distinct metabolism in SARS-CoV2-vaccination- and recall-antigen-specific immunity

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Abstract

Functional avidity is supposed to critically shape the quality of immune responses, thereby impacting host protection against infectious agents including SARS-CoV2. Here we show that after human SARS-CoV2 vaccination, a large portion of high-avidity spike-specific CD4+ T cells lose CD3 expression after in vitro activation. The CD3- subset is enriched for cytokine positive cells, including elevated per-cell expression levels, and shows increased polyfunctionality. Assessment of key metabolic pathways by flow cytometry revealed that superior functionality is accompanied by a shift towards fatty acid-synthesis at the expense of their oxidation, whereas glucose transport and glycolysis were similarly regulated in SARS-CoV2-specific CD3- and CD3+ subsets. As opposed to their CD3+ counterparts, frequencies of vaccine-specific CD3- T cells positively correlate with both the size of the naïve CD4+ T cell pool and vaccine-specific IgG levels. Moreover, their frequencies negatively correlate with advancing age and are impaired in patients under immunosuppressive therapy. Typical recall-antigen-reactive T cells show a comparable segregation into functionally and metabolically distinct CD3+ and CD3- subsets, but are quantitatively maintained upon ageing, likely due to earlier recruitment in life. In summary, our data identify CD3- T helper cells as correlates of high quality immune responses that are impaired in at-risk populations.

Authors

Arne Sattler, Stefanie Gamradt, Vanessa Proß, Linda Marie Laura Thole, An He, Eva Vanessa Schrezenmeier, Katharina Jechow, Stefan M. Gold, Soeren Lukassen, Christian Conrad, Katja Kotsch

BDNF and cAMP are neuroprotective in a porcine model of traumatic optic neuropathy

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Abstract

Traumatic optic neuropathy (TON) is a devastating condition that can occur after blunt or penetrating trauma to the head, leading to visual impairment or blindness. Despite these debilitating effects, no clinically available therapeutic targets neuroprotection or promotes axon regeneration in this or any optic neuropathy. Limited data in large animal models is a major obstacle to advancing treatments toward clinical therapeutics. To address this issue, we refined a surgical model of TON in Yucatan minipigs. First, we validated the model by demonstrating visual impairment by flash visual-evoked potential and retinal ganglion cell degeneration and death. Next, we developed and optimized a delivery method and non-toxic dosing of intravitreal brain-derived neurotrophic factor (BDNF) and cyclic adenosine monophosphate (cAMP). Finally, we showed that intravitreal injection of BDNF and cAMP rescued visual function and protected against retinal ganglion cell death and optic nerve axon degeneration. Together these data in a pre-clinical large animal model advance our understanding of and ability to model TON and further identify and develop candidate clinical therapeutics.

Authors

Kathleen Heng, Brent K. Young, BaoXiang Li, Ashley D. Nies, Xin Xia, Runxia R. Wen, Roopa Dalal, Gregory T. Bramblett, Andrew W. Holt, Jeffery M. Cleland, Jason N. Harris, Albert Y. Wu, Jeffrey L. Goldberg