Periodic Reporting for period 3 - AngioUnrestUHD (Understanding and modulating vascular arrest with ultra-high definition)
Période du rapport: 2024-03-01 au 2025-08-31
Blood vessels are a major player in cancer or cardiovascular disease. Tumors can only develop and spread when blood vessels are formed. The formation of new blood vessels is a process named as angiogenesis, and it supports tumor or tissue growth. In cardiovascular disease or in wounds, the induction of angiogenesis can be beneficial to improve tissue vascularization and function.
Research in the last years identified the major molecular mechanisms controlling angiogenesis, and showed us what to expect from targeting those mechanisms during cancer development or after cardiovascular ischemia. In general, scientists have succeeded in effectively blocking angiogenesis, which has been shown to delay tumor growth and sometimes prevent metastasis. However, inducing effective or productive angiogenesis in ischemic or damaged tissues has been a much more difficult task. An easy to understand comparison is that is much easier to demolish than build a house.
One of the main goals of this ERC Consolidator project is to identify new genetic and pharmacologically targetable mechanisms to induce effective angiogenesis in normal or ischemic/damaged tissues. In the last years scientists identified specific growth or inhibitory factors of angiogenesis. Therefore, adding the identified growth factors or specifically blocking their inhibitors was explored in clinical settings. However, in most cases the results were disappointing. Before the start of this project, our laboratory found that whenever angiogenesis is stimulated above a certain threshold, vessels upregulate the expression of molecules that block their own proliferation. These are called proliferation checkpoints, and are considered to protect cells from DNA damage. In our lab, we knew that vascular cells can proliferate and have a normal function without some of these overprotective checkpoints. Therefore, one of the goals of this project was to test if we could boost therapeutic angiogenesis by preventing the function of some of these proliferation checkpoints.
The second major goal of this project was to understand how vessels form, proliferate and distribute their building blocks across tissues, during organ development, homeostasis or in disease. Our laboratory developed in the last years unique genetic technologies that allow us to follow how every single cell of blood vessels clonally expand and migrate in a mouse. This allowed us to see that each individual vascular cell has unique properties. Some cells clonally expand immensely more than most of their neighbours. We are trying to discover and understand the genetic and epigenetic mechanisms responsible for that cell-to-cell difference in proliferation, in order to find new ways to promote it. We also identified mechanisms responsible for the mobilization of vascular cells across a growing vasculature. Some cells tend to form arteries, whereas others veins, and this can be changed pharmacologically. This will help us to better understand the origin and development of human arteriovenous malformations (AVMs), and also design better therapies to promote the formation of functional blood vessels in disease.
Research in the last years identified the major molecular mechanisms controlling angiogenesis, and showed us what to expect from targeting those mechanisms during cancer development or after cardiovascular ischemia. In general, scientists have succeeded in effectively blocking angiogenesis, which has been shown to delay tumor growth and sometimes prevent metastasis. However, inducing effective or productive angiogenesis in ischemic or damaged tissues has been a much more difficult task. An easy to understand comparison is that is much easier to demolish than build a house.
One of the main goals of this ERC Consolidator project is to identify new genetic and pharmacologically targetable mechanisms to induce effective angiogenesis in normal or ischemic/damaged tissues. In the last years scientists identified specific growth or inhibitory factors of angiogenesis. Therefore, adding the identified growth factors or specifically blocking their inhibitors was explored in clinical settings. However, in most cases the results were disappointing. Before the start of this project, our laboratory found that whenever angiogenesis is stimulated above a certain threshold, vessels upregulate the expression of molecules that block their own proliferation. These are called proliferation checkpoints, and are considered to protect cells from DNA damage. In our lab, we knew that vascular cells can proliferate and have a normal function without some of these overprotective checkpoints. Therefore, one of the goals of this project was to test if we could boost therapeutic angiogenesis by preventing the function of some of these proliferation checkpoints.
The second major goal of this project was to understand how vessels form, proliferate and distribute their building blocks across tissues, during organ development, homeostasis or in disease. Our laboratory developed in the last years unique genetic technologies that allow us to follow how every single cell of blood vessels clonally expand and migrate in a mouse. This allowed us to see that each individual vascular cell has unique properties. Some cells clonally expand immensely more than most of their neighbours. We are trying to discover and understand the genetic and epigenetic mechanisms responsible for that cell-to-cell difference in proliferation, in order to find new ways to promote it. We also identified mechanisms responsible for the mobilization of vascular cells across a growing vasculature. Some cells tend to form arteries, whereas others veins, and this can be changed pharmacologically. This will help us to better understand the origin and development of human arteriovenous malformations (AVMs), and also design better therapies to promote the formation of functional blood vessels in disease.
We started the ERC Consolidator project by analysing the effect of single or combined loss of different genes coding for proteins that induce the arrest of cell proliferation when angiogenesis is stimulated above a certain threshold. This showed that these genes have a minor role and are largely dispensable for physiological angiogenesis, but they are very important for pathological angiogenesis, such as angiogenesis ocurring in tumors, or when angiogenesis is overstimulated by pharmacological compounds.
In a related study we published recently (Fernandez-Chacon et al., 2023 Nature Cardiovascular Research), we found that often vascular transcriptional or angiogenic states do not correlate with the observed vascular pathophysiology. These abnormal transcriptional states are often markers of the vascular malformation and pathology, but these genetic programs do not drive the pathology. We confirmed this when we found that certain pharmacological compounds could induce or reverse those abnormal genetic programs, but not the vascular pathophysiology. These results conceptually changed our understanding of the mechanisms causing the vascular abnormalization and toxicity of certain compounds used in the clinics to deregulate tumor angiogenesis.
One of the difficulties in our field is to determine how single vascular cells clonally expand and mobilize to form vessels over long periods of time. And to interrogate gene function in single cells. With this in mind, we developed recently new genetic tools of broad relevance that enable us to reliably induce any type of genetic mutation in single cells and trace their behaviour over long periods of time. One of these technologies is named as iSuRe-HadCre and allow us to induce and ensure Cre-dependent genetic deletions in single cells expressing a reporter (Garcia-Gonzalez et al., 2024 Nucleic Acids Research) and the other is iFlpMosaics (Garcia-Gonzalez et al., 2025 Nature Methods) and enable us to multispectrally barcode, together with gene-targeted modifications, mutant and wildtype cells in any developing or quiescent tissue. This allowed us to see how specific genes control the clonal expansion and mobilization of single cells across a growing vascular bed, by microscopic imaging and restrospective clonal analysis but also by single cell RNA sequencing (Garcia-Gonzalez and Rocha et al., BioRxiv 2025 and De Andres-Laguillo et al., Biorxiv 2025). In general these technologies allow us to better understand why somatic mutations in some genes, and not others, cause vascular malformations, and what are the mechanisms of long-term resistance to the loss of VEGF signalling.
In a related study we published recently (Fernandez-Chacon et al., 2023 Nature Cardiovascular Research), we found that often vascular transcriptional or angiogenic states do not correlate with the observed vascular pathophysiology. These abnormal transcriptional states are often markers of the vascular malformation and pathology, but these genetic programs do not drive the pathology. We confirmed this when we found that certain pharmacological compounds could induce or reverse those abnormal genetic programs, but not the vascular pathophysiology. These results conceptually changed our understanding of the mechanisms causing the vascular abnormalization and toxicity of certain compounds used in the clinics to deregulate tumor angiogenesis.
One of the difficulties in our field is to determine how single vascular cells clonally expand and mobilize to form vessels over long periods of time. And to interrogate gene function in single cells. With this in mind, we developed recently new genetic tools of broad relevance that enable us to reliably induce any type of genetic mutation in single cells and trace their behaviour over long periods of time. One of these technologies is named as iSuRe-HadCre and allow us to induce and ensure Cre-dependent genetic deletions in single cells expressing a reporter (Garcia-Gonzalez et al., 2024 Nucleic Acids Research) and the other is iFlpMosaics (Garcia-Gonzalez et al., 2025 Nature Methods) and enable us to multispectrally barcode, together with gene-targeted modifications, mutant and wildtype cells in any developing or quiescent tissue. This allowed us to see how specific genes control the clonal expansion and mobilization of single cells across a growing vascular bed, by microscopic imaging and restrospective clonal analysis but also by single cell RNA sequencing (Garcia-Gonzalez and Rocha et al., BioRxiv 2025 and De Andres-Laguillo et al., Biorxiv 2025). In general these technologies allow us to better understand why somatic mutations in some genes, and not others, cause vascular malformations, and what are the mechanisms of long-term resistance to the loss of VEGF signalling.
All results mentioned above constitute a significant advance beyond the state of the art. They either significantly increased our knowledge of important aspects of vascular biology or provide an advancement in the technology used to perform single cell analysis and conditional genetics in mouse models.
Altogether, thanks to this ERC project, we obtained a much deeper understanding of the genetic mechanisms controlling vascular growth/angiogenesis in different physiological and pathological contexts, and also we learned a lot more about how cells form arteries and veins.
In the next stage of the project, we expect to continue to expand and refine our understanding of these mechanisms, but also identify new compounds and therapeutic strategies to promote the effective development of functional blood vessels in disease settings.
We also hope to conclude and publish our findings in several research articles, that will further contribute to the general field effort in understanding the biology of blood vessels and their therapeutic targeting in cardiovascular disease and cancer.
Altogether, thanks to this ERC project, we obtained a much deeper understanding of the genetic mechanisms controlling vascular growth/angiogenesis in different physiological and pathological contexts, and also we learned a lot more about how cells form arteries and veins.
In the next stage of the project, we expect to continue to expand and refine our understanding of these mechanisms, but also identify new compounds and therapeutic strategies to promote the effective development of functional blood vessels in disease settings.
We also hope to conclude and publish our findings in several research articles, that will further contribute to the general field effort in understanding the biology of blood vessels and their therapeutic targeting in cardiovascular disease and cancer.