Angiogenesis
Abstract
The prognosis for colorectal cancer (CRC) patients with liver metastasis remains poor, and the molecular mechanisms driving CRC liver metastasis are still not fully understood. Hypoxia-induced extracellular vesicles (H-EVs) derived from tumors have emerged as key players in inducing angiogenesis by transferring non-coding RNAs. However, the specific role of CRC-derived hypoxic EVs (H-EVs) in regulating the formation of the pre-metastatic microenvironment (PMN) by inducing angiogenesis remains unclear. Our study demonstrates that H-EVs induce both angiogenesis and liver metastasis. Through microRNA microarray analysis, we identified a reduction in miR-6084 levels within H-EVs. We found that miR-6084 inhibits angiogenesis by being transferred to endothelial cells via EVs. In endothelial cells, miR-6084 directly targets ANGPTL4 mRNA, thereby suppressing angiogenesis through ANGPTL4-mediated JAK2/STAT3 pathway. Furthermore, we uncovered that SP1 acts as a transcription factor regulating miR-6084 transcription, while HIF1A decreases miR-6084 expression by promoting SP1 protein dephosphorylation and facilitating ubiquitin‒proteasome degradation in SW620 cells. In clinical samples, we observed low expression of miR-6084 in plasma-derived EVs from CRC patients with liver metastasis. In summary, our findings suggest that CRC-derived hypoxic EVs promote angiogenesis and liver metastasis through the HIF1A/SP1/miR-6084/ANGPTL4 axis. Additionally, miR-6084 holds promise as a potential diagnostic and prognostic biomarker for CRC liver metastasis.
Authors
Yang Zhang, Xuyang Yang, Su Zhang, Qing Huang, Sicheng Liu, Lei Qiu, Mingtian Wei, Xiangbing Deng, Wenjian Meng, Hai-Ning Chen, Yaguang Zhang, Junhong Han, Ziqiang Wang
Abstract
Multiple members of the caveolae-associated protein (Cavin) family are implicated in angiogenesis. However, the specific role of CAVIN3 in pathological angiogenesis within the eye remains unclear. The present study demonstrated that CAVIN3 knockdown in endothelial cells (ECs) promoted vascular normalization in ocular pathological neovascularization. Elevated CAVIN3 expression was observed in the ECs of retinal pigment epithelium/choroid complexes from patients with neovascular age-related macular degeneration and fibrovascular membranes from patients with proliferative diabetic retinopathy. Additionally, upregulated Cavin3 expression was detected in laser-induced choroidal neovascularization (CNV) and oxygen-induced retinopathy (OIR) mouse models. In both OIR and CNV mice, Cavin3 knockdown inhibited pathological neovascularization. Cavin3 deficiency further disrupted EC proliferation and vascular sprouting, thereby promoting vascular normalization by partially restoring microenvironmental hypoxia and reestablishing pericyte-EC interactions. Mechanistically, we demonstrated that zinc finger E-box–binding homeobox 1 (ZEB1) regulated CAVIN3 transcription in ECs under hypoxic conditions. CAVIN3 deficiency modulated pathological vascularization by inhibiting ERK phosphorylation, which downregulated jagged 1 (JAG1) expression. Conclusively, this study elucidated the protective role of endothelial CAVIN3 deficiency in pathological neovascularization models, addressing a gap in understanding the regulatory role of Cavins in angiogenesis. These findings suggested a therapeutic direction for ocular neovascular diseases.
Authors
Weiqi Li, Yeran Zhang, Hongjing Zhu, Na Su, Ruxu Sun, Xiying Mao, Qin Yang, Songtao Yuan
Abstract
Small for gestational age (SGA), with increased risk of adult-onset cardiovascular diseases and metabolic syndromes, is known to associate with endothelial dysfunction, but the pathogenic mechanisms remain unclear. In this study, the pathological state of human umbilical vein endothelial cells (HUVECs) from SGA individuals was characterized by presenting increased angiogenesis, migration, proliferation, and wound healing ability relative to their normal counterparts. Genome-wide mapping of transcriptomes and open chromatins unveiled global gene expression alterations and chromatin remodeling in SGA-HUVECs. Specifically, we revealed increased chromatin accessibility at active enhancers, along with dysregulation of genes associated with angiogenesis, and further identified CD44 as the key gene driving HUVECs’ dysfunction by regulating pro-angiogenic genes’ expression and activating phosphorylated ERK1/2 and phosphorylated endothelial NOS expression in SGA. In SGA-HUVECs, CD44 was abnormally upregulated by 3 active enhancers that displayed increased chromatin accessibility and interacted with CD44 promoter. Subsequent motif analysis uncovered activating protein-1 (AP-1) as a crucial transcription factor regulating CD44 expression by binding to CD44 promoter and associated enhancers. Enhancers CRISPR interference and AP-1 inhibition restored CD44 expression and alleviated the hyperangiogenesis of SGA-HUVECs. Together, our study provides a foundational understanding of the epigenetic alterations driving pathological angiogenesis and offers potential therapeutic insights into addressing endothelial dysfunction in SGA.
Authors
Lingling Yan, Zhimin Zhou, Shengcai Chen, Xin Feng, Junwen Mao, Fang Luo, Jianfang Zhu, Xiuying Chen, Yingying Hu, Yuan Wang, Bingbing Wu, Lizhong Du, Chunlin Wang, Liang Gong, Yanfen Zhu
Abstract
Somatic activating mutations in KRAS can cause complex lymphatic anomalies (CLAs). However, the specific processes that drive KRAS-mediated CLAs have yet to be fully elucidated. Here, we used single-cell RNA sequencing to construct an atlas of normal and KrasG12D-malformed lymphatic vessels. We identified six subtypes of lymphatic endothelial cells (LECs) in the lungs of adult wild-type mice (Ptx3, capillary, collecting, valve, mixed, and proliferating). To determine when the LEC subtypes were specified during development, we integrated our data with data from four stages of development. We found that proliferating and Ptx3 LECs were prevalent during early lymphatic development and that collecting and valve LECs emerged later in development. Additionally, we discovered that the proportion of Ptx3 LECs decreased as the lymphatic network matured but remained high in KrasG12D mice. We also observed that the proportion of collecting and valve LECs was lower in KrasG12D mice than in wild-type mice. Last, we found that immature lymphatic vessels in young mice were more sensitive to the pathologic effects of KrasG12D than mature lymphatic vessels in older mice. Together, our results expand the current model for the development of the lymphatic system and suggest that KRAS mutations impair the maturation of lymphatic vessels.
Authors
Lorenzo M. Fernandes, Danielle Griswold-Wheeler, Jeffrey D. Tresemer, Angelica Vallejo, Neda Vishlaghi, Benjamin Levi, Abigail Shapiro, Joshua P. Scallan, Michael T. Dellinger
Abstract
Diabetes mellitus can cause impaired and delayed wound healing, leading to lower extremity amputations; however, the mechanisms underlying the regulation of vascular endothelial growth factor–dependent (VEGF-dependent) angiogenesis remain unclear. In our study, the molecular underpinnings of endothelial dysfunction in diabetes are investigated, focusing on the roles of disabled-2 (Dab2) and Forkhead box M1 (FOXM1) in VEGF receptor 2 (VEGFR2) signaling and endothelial cell function. Bulk RNA-sequencing analysis identified significant downregulation of Dab2 in high-glucose-treated primary mouse skin endothelial cells. In diabetic mice with endothelial deficiency of Dab2, in vivo and in vitro angiogenesis and wound healing were reduced when compared with wild-type diabetic mice. Restoration of Dab2 expression by injected mRNA-containing, LyP-1–conjugated lipid nanoparticles rescued impaired angiogenesis and wound healing in diabetic mice. Furthermore, FOXM1 was downregulated in skin endothelial cells under high-glucose conditions as determined by RNA-sequencing analysis. FOXM1 was found to bind to the Dab2 promoter, regulating its expression and influencing VEGFR2 signaling. The FOXM1 inhibitor FDI-6 reduced Dab2 expression and phosphorylation of VEGFR2. Our study provides evidence of the crucial roles of Dab2 and FOXM1 in diabetic endothelial dysfunction and establishes targeted delivery as a promising treatment for diabetic vascular complications.
Authors
Sudarshan Bhattacharjee, Jianing Gao, Yao Wei Lu, Shahram Eisa-Beygi, Hao Wu, Kathryn Li, Amy E. Birsner, Scott Wong, Yudong Song, John Y-J. Shyy, Douglas B. Cowan, Wendong Huang, Wenyi Wei, Masanori Aikawa, Jinjun Shi, Hong Chen
Abstract
Macrophage plays a crucial role in promoting perfusion recovery and revascularization after ischemia through anti-inflammatory polarization, a process essential for the treatment of peripheral arterial disease (PAD). Mitochondrial dynamics, particularly regulated by the fission protein DRP1, are closely linked to macrophage metabolism and inflammation. However, the role of DRP1 in reparative neovascularization remains unexplored. Here we show that DRP1 expression was increased in F4/80+ macrophages within ischemic muscle at day 3 after hindlimb ischemia (HLI), an animal model of PAD. Mice lacking Drp1 in myeloid cells exhibited impaired limb perfusion recovery, angiogenesis and muscle regeneration post-HLI. These effects were associated with increased pro-inflammatory M1-like macrophages, p-NFkB and TNFα, and reduced anti-inflammatory M2-like macrophages and p-AMPK in ischemic muscle of myeloid Drp1–/– mice. In vitro, Drp1-deficient macrophages under hypoxia serum starvation (HSS), an in vitro PAD model, demonstrated enhanced glycolysis via reducing p-AMPK as well as mitochondrial dysfunction, and excessive mitochondrial ROS production, resulting in increased pro-inflammatory M1-gene and reduced anti-inflammatory M2-gene expression. Conditioned media from HSS-treated Drp1–/– macrophages exhibited increased pro-inflammatory cytokine secretion, leading to suppressed angiogenesis in endothelial cells. Thus, macrophage DRP1 deficiency under ischemia drives pro-inflammatory metabolic reprogramming and macrophage polarization, limiting revascularization in experimental PAD.
Authors
Shikha Yadav, Vijay C. Ganta, Sudhahar Varadarajan, Vy Ong, Yang Shi, Archita Das, Dipankar Ash, Sheela Nagarkoti, Malgorzata McMenamin, Stephanie Kelley, Tohru Fukai, Masuko Ushio-Fukai
Abstract
Some endothelial cells in the tumor vasculature expressed a system L amino acid transporter LAT1. To elucidate the role of LAT1 in tumor related endothelial cells, tumor cells were injected into endothelial specific LAT1 conditional knockout mice (Slc7a5flox/flox; Cdh5-Cre-ERT2) and found that the shape of the tumor vasculature was normalized and that the size and numbers of lung metastasis was reduced. TNFα-induced expression of VCAM1 and E-selectin at the surface of HUVEC, both of which are responsible for enhanced monocyte attachment and pre-metastatic niche formation, was reduced in the presence of LAT1 inhibitor, nanvuranlat. Deprivation of tryptophan, an LAT1 substrate, mimicked LAT1 inhibition, which led to activation of MEK1/2-ERK1/2 pathway and subsequent cystathionine γ lyase (CTH) induction. Increased production of hydrogen sulfide (H2S) by CTH was at least partially responsible for tumor vascular normalization, leading to decreased leakiness and enhanced delivery of chemotherapeutic agents to the tumor.
Authors
Jun-ichi Suehiro, Toru Kimura, Toshiyuki Fukutomi, Hisamichi Naito, Yasuharu Kanki, Youichiro Wada, Yoshiaki Kubota, Nobuyuki Takakura, Hiroyuki Sakurai
Abstract
RNA-binding proteins (RBPs) interact with RNA and ubiquitously regulate RNA transcripts during their life cycle, playing a fundamental role in the progression of angiogenesis-related diseases. In the skeletal system, endothelium-dependent angiogenesis is indispensable for bone formation. However, the role of RBPs in endothelium-dependent bone formation is unclear. Here, we show that RBP–Y-box-binding protein 1 (YBX1) was strongly reduced in the bone vasculature of ovariectomy (OVX) mice. Endothelial cell–specific deletion of Ybx1 impaired CD31-high, endomucin-high (CD31hiEMCNhi) endothelium morphology and resulted in low bone mass whereas Ybx1 overexpression promoted angiogenesis-dependent osteogenesis and ameliorated bone loss. Mechanistically, YBX1 deletion disrupted CD31, EMCN, and bone morphogenetic protein 4 (BMP4) stability in an m5C-dependent manner and blocked endothelium-derived BMP4 release, thereby inhibiting osteogenic differentiation of bone mesenchymal stromal cells. Administration of recombinant BMP4 protein restored impaired bone formation in Ybx1 deletion mice. Tail vein injection of CD31-modified polyethylene glycol–poly (lactic-co-glycolic acid) carrying sciadopitysin, a natural YBX1 agonist, pharmacologically partially reversed CD31hiEMCNhi vessels’ decline and improved bone mass in both OVX and aging animals. These findings demonstrated the role of RBP-YBX1 in angiogenesis-dependent bone formation and provided a therapeutic approach for ameliorating osteoporosis.
Authors
Yu-Jue Li, Qi Guo, Ming-Sheng Ye, GuangPing Cai, Wen-Feng Xiao, Sheng Deng, Ye Xiao
Abstract
TDP-43 is a DNA/RNA-binding protein that regulates gene expression and its malfunction in neurons has been causally associated with multiple neurodegenerative disorders. Although progress has been made in understanding the functions of TDP-43 in neurons, little is known about its role in endothelial cells (ECs), angiogenesis and vascular function. Using inducible EC-specific TDP-43 knockout mice, we showed that TDP-43 is required for sprouting angiogenesis, vascular barrier integrity and blood vessel stability. Postnatal EC-specific deletion of TDP-43 leaded to retinal hypovascularization due to defects in vessel sprouting associated with reduced EC proliferation and migration. In mature blood vessels, loss of TDP-43 disrupted the blood-brain barrier and triggered vascular degeneration. These vascular defects were associated with an inflammatory response in the central-nervous system with activation of microglia and astrocytes. Mechanistically, deletion of TDP-43 disrupted fibronectin matrix around sprouting vessels and reduced -catenin signaling in ECs. Together, our results indicate that TDP-43 is essential for the formation of a stable and mature vasculature.
Authors
Víctor Arribas, Yara Onetti, Marina Ramiro-Pareta, Pilar Villacampa, Heike Beck, Mariona Alberola, Anna Esteve-Codina, Angelika Merkel, Markus Sperandio, Ofelia M. Martínez-Estrada, Bettina Schmid, Eloi Montanez
Abstract
We previously showed that ablation of tumor hypoxia can sensitize tumors to immune checkpoint blockade (ICB). Here, we used a Kras+/G12DTP53+/R172HPdx1-Cre (KPC) derived model of pancreatic adenocarcinoma (PDAC) to examine the tumor response and adaptive resistance mechanisms involved in response to two established methods of hypoxia-reducing therapy: the hypoxia-activated prodrug TH-302 and vascular endothelial growth factor receptor 2 (VEGFR-2) blockade. The combination of both modalities normalized tumor vasculature, increased DNA damage and cell death, and delayed tumor growth. In contrast to prior cancer models, the combination did not alleviate overall tissue hypoxia or sensitize these KPC tumors to ICB therapy despite qualitative improvements to the CD8 T cell response. Bulk-tumor RNA sequencing, flow cytometry, and adoptive myeloid cell transfer suggested that treated tumor cells increased their capacity to recruit granulocytic myeloid derived suppressor cells (G-MDSC) through CCL9 secretion. Blockade of the CCL9-CCR1 axis could limit G-MDSC migration, and depletion of Ly6G-positive cells could sensitize tumors to the combination of TH-302 and anti-VEGFR-2 with ICB. Together, these data suggest that pancreatic tumors modulate G-MDSC migration as an adaptive response to vascular normalization, and that these immunosuppressive myeloid cells act in a setting of persistent hypoxia to maintain adaptive immune resistance.
Authors
Arthur Liu, Seth T. Gammon, Federica Pisaneschi, Akash Boda, Casey R. Ager, David Piwnica-Worms, David S. Hong, Michael A. Curran
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