Early Detection and Staging of Lung Fibrosis Enabled by Collagen-Targeted MRI Protein Contrast Agent.

Chronic lung diseases, such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD), are major leading causes of death worldwide and are generally associated with poor prognoses. The heterogeneous distribution of collagen, mainly type I collagen associated with excessive collagen deposition, plays a pivotal role in the progressive remodeling of the lung parenchyma to chronic exertional dyspnea for both IPF and COPD. To address the pressing need for noninvasive early diagnosis and drug treatment monitoring of pulmonary fibrosis, we report the development of human collagen-targeted protein MRI contrast agent (hProCA32.collagen) to specifically bind to collagen I overexpressed in multiple lung diseases. When compared to clinically approved Gd3+ contrast agents, hProCA32.collagen exhibits significantly better r1 and r2 relaxivity values, strong metal binding affinity and selectivity, and transmetalation resistance. Here, we report the robust detection of early and late-stage lung fibrosis with stage-dependent MRI signal-to-noise ratio (SNR) increase, with good sensitivity and specificity, using a progressive bleomycin-induced IPF mouse model. Spatial heterogeneous mapping of usual interstitial pneumonia (UIP) patterns with key features closely mimicking human IPF, including cystic clustering, honeycombing, and traction bronchiectasis, were noninvasively detected by multiple MR imaging techniques and verified by histological correlation. We further report the detection of fibrosis in the lung airway of an electronic cigarette-induced COPD mouse model, using hProCA32.collagen-enabled precision MRI (pMRI), and validated by histological analysis. The developed hProCA32.collagen is expected to have strong translational potential for the noninvasive detection and staging of lung diseases, and facilitating effective treatment to halt further chronic lung disease progression.

IKKß Inhibition Attenuates Epithelial Mesenchymal Transition of Human Stem Cell-Derived Retinal Pigment Epithelium.

Epithelial-mesenchymal transition (EMT), which is well known for its role in embryonic development, malignant transformation, and tumor progression, has also been implicated in a variety of retinal diseases, including proliferative vitreoretinopathy (PVR), age-related macular degeneration (AMD), and diabetic retinopathy. EMT of the retinal pigment epithelium (RPE), although important in the pathogenesis of these retinal conditions, is not well understood at the molecular level. We and others have shown that a variety of molecules, including the co-treatment of human stem cell-derived RPE monolayer cultures with transforming growth factor beta (TGF-ß) and the inflammatory cytokine tumor necrosis factor alpha (TNF-α), can induce RPE-EMT; however, small molecule inhibitors of RPE-EMT have been less well studied. Here, we demonstrate that BAY651942, a small molecule inhibitor of nuclear factor kapa-B kinase subunit beta (IKKß) that selectively targets NF-κB signaling, can modulate TGF-ß/TNF-α-induced RPE-EMT. Next, we performed RNA-seq studies on BAY651942 treated hRPE monolayers to dissect altered biological pathways and signaling events. Further, we validated the effect of IKKß inhibition on RPE-EMT-associated factors using a second IKKß inhibitor, BMS345541, with RPE monolayers derived from an independent stem cell line. Our data highlights the fact that pharmacological inhibition of RPE-EMT restores RPE identity and may provide a promising approach for treating retinal diseases that involve RPE dedifferentiation and EMT.

Mitophagy initiates retrograde mitochondrial-nuclear signaling to guide retinal pigment cell heterogeneity.

Age-related macular degeneration (AMD), the leading cause of blindness among the elderly, is without treatment for early disease. Degenerative retinal pigment epithelial (RPE) cell heterogeneity is a well-recognized but understudied pathogenic factor. Due to the daily phagocytosis of photoreceptor outer segments, unique photo-oxidative stress, and high metabolism for maintaining vision, the RPE has robust macroautophagy/autophagy, and mitochondrial and antioxidant networks. However, the autophagy subtype, mitophagy, in the RPE and AMD is understudied. Here, we found decreased PINK1 (PTEN induced kinase 1) in perifoveal RPE of early AMD eyes. PINK1-deficient RPE have impaired mitophagy and mitochondrial function that triggers death-resistant epithelial-mesenchymal transition (EMT). This reprogramming is mediated by novel retrograde mitochondrial-nuclear signaling (RMNS) through superoxide, NFE2L2 (NFE2 like bZIP transcription factor 2), TXNRD1 (thioredoxin reductase 1), and phosphoinositide 3-kinase (PI3K)-AKT (AKT serine/threonine kinase) that induced canonical transcription factors ZEB1 (zinc finger E-box binding homeobox 1) and SNAI1 (Snail family transcriptional repressor 1) and an EMT transcriptome. NFE2L2 deficiency disrupted RMNS that paradoxically normalized morphology but decreased function and viability. Thus, RPE heterogeneity is defined by the interaction of two cytoprotective pathways that is triggered by mitophagy function. By neutralizing the consequences of impaired mitophagy, an antioxidant dendrimer tropic for the RPE and mitochondria, EMT (a recognized AMD alteration) was abrogated to offer potential therapy for early AMD, a stage without treatment.Abbreviations: ACTB: actin beta; AKT: AKT serine/threonine kinase; AMD: age-related macular degeneration; CCCP: cyanide m-chlorophenyl hydrazone; CDH1: cadherin 1; DAVID: Database for Annotation, Visualization and Integrated Discovery; DHE: dihydroethidium; D-NAC: N-acetyl-l-cysteine conjugated to a poly(amido amine) dendrimer; ECAR: extracellular acidification rate; EMT: epithelial-mesenchymal transition; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSEA: Gene Set Enrichment Analysis; HSPD1: heat shock protein family D (Hsp60) member 1; IVT: intravitreal; KD: knockdown; LMNA, lamin A/C; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MMP: mitochondrial membrane potential; NAC: N-acetyl-l-cysteine; NQO1: NAD(P)H quinone dehydrogenase 1; NFE2L2: NFE2 like bZIP transcription factor 2; O2-: superoxide anion; OCR: oxygen consumption rate; PI3K: phosphoinositide 3-kinase; PINK1: PTEN induced kinase 1; RMNS: retrograde mitochondrial-nuclear signaling; ROS: reactive oxygen species; RPE: retinal pigment epithelium; SNAI1: snail family transcriptional repressor 1; TJP1: tight junction protein 1; TPP-D-NAC: triphenyl phosphinium and N-acetyl-l-cysteine conjugated to a poly(amido amine) dendrimer; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20; Trig: trigonelline; TXNRD1: thioredoxin reductase 1; VIM: vimentin; WT: wild-type; ZEB1: zinc finger E-box binding homeobox 1.

Endothelial Autophagy in Coronary Microvascular Dysfunction and Cardiovascular Disease.

Coronary microvascular dysfunction (CMD) refers to a subset of structural and/or functional disorders of coronary microcirculation that lead to impaired coronary blood flow and eventually myocardial ischemia. Amid the growing knowledge of the pathophysiological mechanisms and the development of advanced tools for assessment, CMD has emerged as a prevalent cause of a broad spectrum of cardiovascular diseases (CVDs), including obstructive and nonobstructive coronary artery disease, diabetic cardiomyopathy, and heart failure with preserved ejection fraction. Of note, the endothelium exerts vital functions in regulating coronary microvascular and cardiac function. Importantly, insufficient or uncontrolled activation of endothelial autophagy facilitates the pathogenesis of CMD in diverse CVDs. Here, we review the progress in understanding the pathophysiological mechanisms of autophagy in coronary endothelial cells and discuss their potential role in CMD and CVDs.

P68 RNA Helicase facilitates Breast Cancer progression by promoting Proliferation and Migration via PDGFR-ß/AR axis.

Aberrant expression of P68 RNA helicase (p68), a prototypical member of the DEAD box family of RNA helicases, contributes to tumor development and progression. P68 tyrosine phosphorylation induced by PDGF signaling facilitates cancer metastasis by promoting EMT. In this report, we show that p68 promotes breast cancer cell EMT and cell migration by upregulation of PDGF receptor ß (PDGFR-ß). Knockdown of p68 in MDA-MB-231 and BT549 cells significantly decreases PDGFR-ß both in mRNA and protein levels. P68 promotes EMT and cell migration in response to PDGF-BB stimulation via upregulation of PDGFR-ß, suggesting that p68 enhances PDGF signaling by a positive feedback loop in cancer cells. Furthermore, our study reveals that p68 mediates the effects of PDGFR-ß in regulation of androgen receptor (AR) in breast cancer cells. We demonstrate that p68 and PDGFR-ß co-regulate AR expression and promote androgen-mediated proliferation in breast cancer cells. Our studies uncover an important pathway of p68-PDGFR-ß axis in promoting breast cancer progression.

Pyruvate kinase M2 regulates fibrosis development and progression by controlling glycine auxotrophy in myofibroblasts.

Rationale: Fibrosis is a pathologic condition of abnormal accumulation of collagen fibrils. Collagen is a major extracellular matrix (ECM) protein synthesized and secreted by myofibroblasts, composing mainly (Gly-X-Y)n triplet repeats with >30% Gly residue. During fibrosis progression, myofibroblasts must upregulate glycine metabolism to meet the high demands of amino acids for collagen synthesis. Method: Expression of PKM2 in myofibroblasts was analyzed in cultured fibroblasts and fibrosis disease tissues. Functional roles of PKM2 and PKM2 activator in biosynthesis of serine → glycine and production of collagen from glycolysis intermediates were assayed in cultured activated LX-2 and human primary lung fibroblast cells. Mouse models of Liver, lung, and pancreas fibrosis were employed to analyze treatment effects of PKM2 activator in organ tissue fibrosis. Results: We report here that myofibroblast differentiation upregulates pyruvate kinase M2 (PKM2) and promotes dimerization of PKM2. Dimer PKM2 slows the flow rate of glycolysis and channels glycolytic intermediates to de novo glycine synthesis, which facilitates collagen synthesis and secretion in myofibroblasts. Our results show that PKM2 activator that converts PKM2 dimer to tetramer, inhibits fibrosis progression in mouse models of liver, lung, and pancreatic fibrosis. Furthermore, metabolism alteration by dimer PKM2 increases NADPH production, which consequently protects myofibroblasts from apoptosis. Conclusion: Our study uncovers a novel role of PKM2 in tissue/organ fibrosis, suggesting a possible strategy for treatment of fibrotic diseases using PKM2 activator.

Targeting integrin αvß3 by a rationally designed protein for chronic liver disease treatment.

Chronic Liver Diseases (CLD) are characterized by abnormal accumulation of collagen fibrils, neo-angiogenesis, and sinusoidal remodeling. Collagen deposition along with intrahepatic angiogenesis and sinusoidal remodeling alters sinusoid structure resulting in portal hypertension, liver failure, and other complications. Efforts were made to develop treatments for CLDs. However, the success of such treatments is limited and unpredictable. We report a strategy for CLD treatment by induction of integrin αvß3 mediated cell apoptosis using a rationally designed protein (ProAgio). ProAgio is designed to target integrin αvß3 at a novel site. Integrin αvß3 is highly expressed in activated Hepatic Stellate Cells (HSC), angiogenic endothelium, and capillarized Liver Sinusoidal Endothelial Cells (LSEC). ProAgio induces apoptosis of these disease causative cells. Tests with liver fibrosis mouse models demonstrate that ProAgio reverses liver fibrosis and relieves blood flow resistance by depleting activated HSC and capillarized LSEC. Our studies demonstrate an effective approach for CLD treatment.

Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer.

Oncogenic KRAS mutations combined with the loss of the LKB1 tumor-suppressor gene (KL) are strongly associated with aggressive forms of lung cancer. N6-methyladenosine (m6A) in mRNA is a crucial epigenetic modification that controls cancer self-renewal and progression. However, the regulation and role of m6A modification in this cancer are unclear. We found that decreased m6A levels correlated with the disease progression and poor survival for KL patients. The correlation was mediated by a special increase in ALKBH5 (AlkB family member 5) levels, an m6A demethylase. ALKBH5 gain- or loss-of function could effectively reverse LKB1 regulated cell proliferation, colony formation, and migration of KRAS-mutated lung cancer cells. Mechanistically, LKB1 loss upregulated ALKBH5 expression by DNA hypermethylation of the CTCF-binding motif on the ALKBH5 promoter, which inhibited CTCF binding but enhanced histone modifications, including H3K4me3, H3K9ac, and H3K27ac. This effect could successfully be rescued by LKB1 expression. ALKBH5 demethylation of m6A stabilized oncogenic drivers, such as SOX2, SMAD7, and MYC, through a pathway dependent on YTHDF2, an m6A reader protein. The above findings were confirmed in clinical KRAS-mutated lung cancer patients. We conclude that loss of LKB1 promotes ALKBH5 transcription by a DNA methylation mechanism, reduces m6A modification, and increases the stability of m6A target oncogenes, thus contributing to aggressive phenotypes of KRAS-mutated lung cancer.

Simultaneously targeting cancer-associated fibroblasts and angiogenic vessel as a treatment for TNBC.

Fibrotic tumor stroma plays an important role in facilitating triple-negative breast cancer (TNBC) progression and chemotherapeutic resistance. We previously reported a rationally designed protein (ProAgio) that targets integrin αvß3 at a novel site. ProAgio induces apoptosis via the integrin. Cancer-associated fibroblasts (CAFs) and angiogenic endothelial cells (aECs) in TNBC tumor express high levels of integrin αvß3. ProAgio effectively induces apoptosis in CAFs and aECs. The depletion of CAFs by ProAgio reduces intratumoral collagen and decreases growth factors released from CAFs in the tumor, resulting in decreased cancer cell proliferation and apoptotic resistance. ProAgio also eliminates leaky tumor angiogenic vessels, which consequently reduces tumor hypoxia and improves drug delivery. The depletion of CAFs and reduction in hypoxia by ProAgio decreases lysyl oxidase (LOX) secretion, which may play a role in the reduction of metastasis. ProAgio stand-alone or in combination with a chemotherapeutic agent provides survival benefit in TNBC murine models, highlighting the therapeutic potential of ProAgio as a treatment strategy.