Our laboratory focuses on studying how metabolism, redox signaling, and physical forces regulates vascular homeostasis in development as well as during cancer progression. To accomplish this we aim to take advantage of innovative genetic and imaging technologies as well as new molecular and metabolic approaches in vertebrate models, such as zebrafish and mouse.
In detail, we are currently performing four main projects:
1) Role of isoprenoid metabolism in developmental and disease angiogenesis;
2) Control of protein stabilization in breast cancer progression by UBIAD1 and oxidative stress;
3) Hemodynamic control of vascular maturation via redox metabolism;
4) Role of circadian clock and light in angiogenesis.
- Role of the Isoprenoid Pathway in Angiogenesis:
Although the role of glucose metabolism in angiogenesis is becoming clear, little is still known about lipids and lipid metabolic pathways that are crucial for endothelial cells (ECs) and vascular homeostasis. The isoprenoid pathway serves to produce dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP), which are mainly known for terpenoid biosynthesis in cell membrane maintenance, hormones, protein anchoring, and N-glycosylation. Alternative functions for IPP have not been identified so far. Although some evidences suggests that the block of isoprenoid pathway might impact the endothelial functions in vitro the critical role of this metabolic pathway in vivo angiogenesis has not yet been addressed. In this project we are investigating the role of the isoprenoid metabolic pathway in developing and tumor angiogenesis. By using CRISPR/cas9 technology in zebrafish we have generated genetic mutants of 5 different enzymes of the isoprenoid pathways. The fdps and idi1 mutants show strong vascular phenotypes. We also performed metabolic measurements of isoprenoids in these mutants with interesting results. In endothelial cells isoprenoid blockade increase IPP levels and promote DNA damage. response and Golgi morphology. We are currently investigating the mechanism responsible for these cellular phenotypes. In a parallel, we are also characterizing an endothelial specific cKO mice for idi1. The plan is to perform tumor angiogenesis assays in this conditional line. Our data might offer a solid rationale to screen for new isoprenoid pathway inhibitors, ultimately offering the possibility for industrial valorization.
- A critical function for UBIAD1 and oxidative stress in Breast tumor progression:
UBIAD1 was identified by our laboratory as a non-mitochondrial prenyltransferase that synthesizes CoQ10 in the Golgi membrane compartment. By doing so we demonstrated that UBIAD1 is a critical antioxidant enzymes for the cardiovascular system during its development. Although the biochemical role of UBIAD1 is known, its function in cancer has not yet been investigated. It is now becoming clear that oxidative stress inhibits distant metastasis by melanoma cells while antioxidant promotes melanoma progression. Here we address the role of UBIAD1in regulating tumor progression by regulating oxidative stress. By testing expression of Ubiad1 on different types of human cancers we found that Ubiad1 protein, but not RNA, is upregulated in almost 50% of colon and breast cancer samples, but not in their normal counterparts. More importantly, our analyses also show that Ubiad1 levels is strongly increase in 75% of metastatic breast cancer. To further analyze the role of Ubiad1 in breast cancer initiation and progression we have generated a knock-out mouse for Ubiad1 and cross it with different mice breast cancer models (e.g. erbB2-Neu, PyMT-MMTV). We found that Ubiad1 loss protect from tumor dissemination and lung metastasis in these breast cancer models. Similar results were obtained when in xenograft tumor models (B16 melanoma cells and E0071 breast carcinoma cells silenced or overexpressing UBIAD1). These preliminary results support a critical role for Ubiad1 in maintenance of tumor cell antioxidant defenses during tumor initiation and dissemination. We are currently investigating a novel molecular mechanism responsible for the stabilization of Ubiad1 by oxidative stress in cancer progression (redoxome approach in breast cancer tissues). If true, this idea will provide ground for new therapies against tumor metastasis: as most breast cancer patients die due to the metastasis and not the primary tumor, we believe that targeting Ubiad1 or CoQ10 synthesis would be valuable for the survival of these patients. Furthermore, the highly metastatic, triple negative breast cancers do not respond effectively to the available treatments indicating that anti UBIAD1 inhibition could be a life-saving therapy for these patients.
- Hemodynamic control of vascular maturation via redox metabolism:
Laminar shear stress (LSS), ROS signaling and cell metabolism are known to play independently play crucial roles in controlling endothelial cells (EC) function and homeostasis. Interestingly, studies have demonstrated that LSS can regulate ROS production and signaling. On the other hand, metabolic pathways can regulate angiogenesis, such as glicolysis and others. Among the most important but yet uncharacterized metabolic pathway in ECs we have the oxidative pentose phosphate pathway (oxPPP). oxPPP is composed of two major enzymes, G6PD and PGD, that are able to control oxidative stress protection and nucleic acid synthesis in different cell types. However, the role of oxPPP in angiogenesis is still unclear. Also whether, LSS influence oxPPP or whether ROS can target this metabolic pathway is unknown. Interestingly, LSS and oxPPP acts via the regulation of ROS synthesis and homeostasis. ROS play a key role in cell signaling through cysteine oxidative post-translational modification (Ox-PTM) of selective target proteins. Despite the described role of the PPP and LSS in EC function is documented, no study has yet determined the relation between both LSS, ROS and oxPPP in the control of EC function. Particularly, nothing is known about how these events are connected and whether they might control epigenetic modification and gene expression. This project aims to establish a LSS®ROS®oxPPP signaling that may be involved in the control of vascular development and mural cells function by epigenetic events. We have recently performed a redox proteomic screen (OxICAT) to identify sensitive redox cysteine and to target enzymes during LSS. We identified 1524 peptides and 978 proteins exhibiting cysteine Ox-PTM during LSS including those involved in cytoskeleton, translation or cell cycle protein. We also found that LSS-mediated ROS production can oxidase the rate-limiting enzyme of the oxPPP, PGD, on a specific cysteine residue (C289A). The effect of this Ox-PTM on PGD activity as well as what its molecular and biological function is unknown. We have some preliminary results in zebrafish from testing endothelial specific embryos for PGD and G6PD (conditional CRISPR/Cas9 technology) that found the deletion of oxPPP in ECs in vivo can lead to an impairment of vascular mural cells recruitment. To further explore how EC may control vascular development and function via blood flow and cell metabolism adaptation through Ox-PTM we have already generated an endothelial-specific PGD mutant mice (EC-PGD-) and a conditional KI mouse (cKI) harbouring a cys289Ala mutation. These conditional allele mice have been crossed with Ve-Cadherin-CreERT2 mice. We are currently verifying the inactivation of PGD specifically in EC by performing gene expression on EC isolated from WT and EC-PGD-/- tissue (e.g. heart, aorta, kidney) and by performing co-immunostaining on the aortic ring (utilizing PGD and PECAM-1 or α-SMA antibodies, specific, respectively, for EC and smooth muscle cells). This deletion approach is able to confirming that the critical role of PGD in endothelial development and function and will open the way to investigating epigenetic events driven by LSS, ROS and metabolism.
- Role of circadian clock and light in angiogenesis:
Mechanisms underlying circadian- and light-regulated physiological processes remain largely unknown. In mammals and other vertebrates, the circadian clock plays a central role in the regulation of physiological functions of almost all tissues and organs. It is understood that some crucial genes involved in the regulation of cell growth, differentiation, and metabolism are tightly regulated by the clock genes. Recent findings indicate that also the UBIAD1 gene is regulated by circadian clocks. Due to the key importance of the circadian clock, certain transcription factors, such as Bmal1 and Clock, and transcription coregulators Period2 and Cryptochrome, or their homologs specialized for circadian regulation of targeted genes, are widely present in vertebrates, including zebrafish. Despite the vast knowledge of circadian biology and angiogenesis, the role of the circadian clock in angiogenesis and vascular patterning remains poorly investigated. Here we plan to define mechanistic insight on the role of the circadian clock and light in regulation of developmental angiogenesis. We show that disruption of the circadian clock by constant exposure to light or genetic manipulation of key genes such as Bmal1 and per2 in zebrafish leads to impaired developmental angiogenesis. A bmal1-specific CRISPR/Cas9 zebrafish mutant show block of sprouting angiogenesis without causing obvious nonvascular phenotypes. Using a Dll4 promoter-reporter system consisting of various deleted promoter mutants, we can show that Bmal1 directly binds to and activates the Dll4 promoter via E-boxes. To further explore how Bmal1 may control vascular development and function via Notch we have already crossed conditional allele of Bmal1 with Ve-Cadherin-CreERT2 mice. We are currently characterizing these mice for developmental angiogenesis (retina angiogenesis) and pathological angiogenesis (tumor xenografts). Additionally, we are also performing ChipSeq experiments in HUVEC with Bmal1 to identify novel Bmal1 target genes in ECs that can explain the angiogenic phenotype in vivo. Such findings may open up new therapeutic approaches in pathological angiogenesis and reasonably extended to other types of vascular-related conditions by targeting the circadian clock.
In summary, our goal is to expand the current vision of endothelial biology by identifying novel metabolic and redox signaling mechanisms and used them to develop novel therapies to treat cancer progression and vascular-related pathologies.
