Periodic Reporting for period 5 - Damocles (Modelling brain aneurysm to elucidate the role of platelets)
Periodo di rendicontazione: 2024-06-01 al 2025-08-31
Intracranial aneurysms (IA), with or without subarachnoid hemorrhage (SAH), remain a major clinical challenge. IA affect about 3% of the population and carry an annual rupture risk of ~1%. When rupture occurs, it leads to SAH, a devastating condition associated with mortality rates of 50–60%. Despite this high burden, current treatments are limited to invasive surgical or endovascular procedures. No pharmacological therapy is currently available to prevent aneurysm growth or rupture, largely due to an incomplete understanding of the underlying mechanisms.
Increasing evidence suggests that inflammation plays a central role in IA pathophysiology. Human aneurysm samples show inflammatory cell infiltration and extensive vascular wall remodeling. Intraluminal thrombus formation is also frequently observed, suggesting that platelets may contribute to disease progression beyond their classical role in hemostasis. Because platelets interact with immune cells and release inflammatory mediators, we hypothesized that platelet signaling pathways contribute to aneurysm formation, progression, and rupture.
To address this hypothesis, we designed an interdisciplinary program combining clinical investigations, biophysical approaches, pharmacology, and in vivo models.
Aim 1 was to characterize platelet activation in patients with IA. We analyzed platelet activation markers in peripheral blood and directly within the aneurysmal sac of patients undergoing surgery for unruptured aneurysms. These studies revealed systemic platelet activation in IA patients and increased inflammatory mediators within the aneurysm lumen, supporting the idea that platelets contribute to the inflammatory microenvironment of aneurysms.
Aim 2 investigated platelet activation under aneurysm-like hemodynamic conditions using artificial vessel systems. These experiments allowed us to study how altered flow patterns and vascular geometry influence platelet activation and platelet–immune cell interactions.
Aim 3 examined platelet signaling pathways in vivo using mouse models of intracranial aneurysm and evaluated the impact of modulating platelet pathways on aneurysm development and rupture.
Conclusions.
Overall, the project demonstrates that platelets actively contribute to intracranial aneurysm pathophysiology beyond thrombosis, particularly through interactions with inflammatory cells such as neutrophils. Our findings identify platelet-driven inflammatory signaling as an important regulator of aneurysm progression and stability, providing a conceptual framework for the development of pharmacological strategies aimed at stabilizing aneurysms and preventing rupture.
Increasing evidence suggests that inflammation plays a central role in IA pathophysiology. Human aneurysm samples show inflammatory cell infiltration and extensive vascular wall remodeling. Intraluminal thrombus formation is also frequently observed, suggesting that platelets may contribute to disease progression beyond their classical role in hemostasis. Because platelets interact with immune cells and release inflammatory mediators, we hypothesized that platelet signaling pathways contribute to aneurysm formation, progression, and rupture.
To address this hypothesis, we designed an interdisciplinary program combining clinical investigations, biophysical approaches, pharmacology, and in vivo models.
Aim 1 was to characterize platelet activation in patients with IA. We analyzed platelet activation markers in peripheral blood and directly within the aneurysmal sac of patients undergoing surgery for unruptured aneurysms. These studies revealed systemic platelet activation in IA patients and increased inflammatory mediators within the aneurysm lumen, supporting the idea that platelets contribute to the inflammatory microenvironment of aneurysms.
Aim 2 investigated platelet activation under aneurysm-like hemodynamic conditions using artificial vessel systems. These experiments allowed us to study how altered flow patterns and vascular geometry influence platelet activation and platelet–immune cell interactions.
Aim 3 examined platelet signaling pathways in vivo using mouse models of intracranial aneurysm and evaluated the impact of modulating platelet pathways on aneurysm development and rupture.
Conclusions.
Overall, the project demonstrates that platelets actively contribute to intracranial aneurysm pathophysiology beyond thrombosis, particularly through interactions with inflammatory cells such as neutrophils. Our findings identify platelet-driven inflammatory signaling as an important regulator of aneurysm progression and stability, providing a conceptual framework for the development of pharmacological strategies aimed at stabilizing aneurysms and preventing rupture.
Since the beginning of the project, we implemented an integrated research strategy combining clinical studies, in vitro approaches, and in vivo models to investigate the role of platelet signaling in intracranial aneurysm (IA) formation, progression, and rupture.
First, we conducted a clinical study with the Department of Neurovascular Surgery at Fondation Rothschild to assess platelet activation in IA patients. Blood samples were collected from the aneurysm sac, the parent artery, and peripheral circulation in patients undergoing surgery for unruptured aneurysms (n>270). Flow cytometry and ELISA analyses revealed increased platelet activation, with elevated P-selectin expression in both peripheral and intracranial samples. Neutrophil-associated inflammatory mediators, including MPO and MMP-9, were also enriched within the aneurysm lumen, indicating a pro-inflammatory microenvironment.
Second, we developed experimental systems mimicking aneurysm-like vascular conditions to study platelet activation and platelet–immune cell interactions under controlled hemodynamic conditions.
Third, we established complementary mouse models of intracranial aneurysm to investigate platelet mechanisms in vivo. We developed a non-invasive transcranial Doppler ultrasound approach to monitor aneurysm progression and a standardized histological scoring system to quantify aneurysm severity.
Using these models, we showed that platelet recruitment to the aneurysmal wall correlates with lesion severity and occurs even in the absence of luminal thrombosis. We also observed systemic platelet pre-activation and increased platelet–neutrophil aggregates, highlighting platelet–neutrophil interactions as drivers of aneurysm inflammation.
Mechanistic studies revealed that platelet receptors PAR4, P2Y12, and GPVI modulate aneurysm severity and inflammatory cell recruitment, while platelet factor 4 (PF4) regulates neutrophil recruitment and aneurysm rupture.
The results have been disseminated through publications and conferences, and led to the filing of two patents targeting platelet pathways to prevent intracranial aneurysm rupture.
First, we conducted a clinical study with the Department of Neurovascular Surgery at Fondation Rothschild to assess platelet activation in IA patients. Blood samples were collected from the aneurysm sac, the parent artery, and peripheral circulation in patients undergoing surgery for unruptured aneurysms (n>270). Flow cytometry and ELISA analyses revealed increased platelet activation, with elevated P-selectin expression in both peripheral and intracranial samples. Neutrophil-associated inflammatory mediators, including MPO and MMP-9, were also enriched within the aneurysm lumen, indicating a pro-inflammatory microenvironment.
Second, we developed experimental systems mimicking aneurysm-like vascular conditions to study platelet activation and platelet–immune cell interactions under controlled hemodynamic conditions.
Third, we established complementary mouse models of intracranial aneurysm to investigate platelet mechanisms in vivo. We developed a non-invasive transcranial Doppler ultrasound approach to monitor aneurysm progression and a standardized histological scoring system to quantify aneurysm severity.
Using these models, we showed that platelet recruitment to the aneurysmal wall correlates with lesion severity and occurs even in the absence of luminal thrombosis. We also observed systemic platelet pre-activation and increased platelet–neutrophil aggregates, highlighting platelet–neutrophil interactions as drivers of aneurysm inflammation.
Mechanistic studies revealed that platelet receptors PAR4, P2Y12, and GPVI modulate aneurysm severity and inflammatory cell recruitment, while platelet factor 4 (PF4) regulates neutrophil recruitment and aneurysm rupture.
The results have been disseminated through publications and conferences, and led to the filing of two patents targeting platelet pathways to prevent intracranial aneurysm rupture.
At the start of this project, intracranial aneurysm (IA) pathophysiology was mainly attributed to altered hemodynamics and chronic inflammation of the arterial wall. Although inflammatory cell infiltration had been described, the role of platelets in aneurysm formation and rupture remained largely unexplored, and platelets were mainly considered in the context of thrombosis after aneurysm rupture.
This project demonstrates that platelets actively contribute to IA pathophysiology. By combining analyses in patients, in vitro experimental systems, and in vivo mouse models, we show that platelets are systemically activated in IA and are recruited to the aneurysmal wall even in the absence of luminal thrombosis. Our results further identify platelet–neutrophil interactions as an important mechanism linking platelet activation to vascular inflammation and aneurysm progression.
We also identified platelet signaling pathways that influence aneurysm severity and stability. Using genetic and pharmacological approaches, we show that platelet receptors such as PAR4 and P2Y12 modulate inflammatory cell recruitment and aneurysm development, while GPVI plays a deleterious role in disease progression. In addition, platelet factor 4 (PF4) was identified as a regulator of neutrophil recruitment and aneurysm rupture, highlighting platelet-derived mediators as previously unrecognized regulators of aneurysm stability.
The project also generated methodological advances, including a non-invasive transcranial Doppler ultrasound approach to monitor aneurysm progression in experimental models and a standardized histological scoring system to quantify aneurysm severity.
Overall, these results expand the current understanding of intracranial aneurysm disease and identify platelet-driven inflammatory pathways as potential therapeutic targets. Ongoing work focuses on disseminating these findings through publications and developing translational applications, supported by patents filed during the project.
This project demonstrates that platelets actively contribute to IA pathophysiology. By combining analyses in patients, in vitro experimental systems, and in vivo mouse models, we show that platelets are systemically activated in IA and are recruited to the aneurysmal wall even in the absence of luminal thrombosis. Our results further identify platelet–neutrophil interactions as an important mechanism linking platelet activation to vascular inflammation and aneurysm progression.
We also identified platelet signaling pathways that influence aneurysm severity and stability. Using genetic and pharmacological approaches, we show that platelet receptors such as PAR4 and P2Y12 modulate inflammatory cell recruitment and aneurysm development, while GPVI plays a deleterious role in disease progression. In addition, platelet factor 4 (PF4) was identified as a regulator of neutrophil recruitment and aneurysm rupture, highlighting platelet-derived mediators as previously unrecognized regulators of aneurysm stability.
The project also generated methodological advances, including a non-invasive transcranial Doppler ultrasound approach to monitor aneurysm progression in experimental models and a standardized histological scoring system to quantify aneurysm severity.
Overall, these results expand the current understanding of intracranial aneurysm disease and identify platelet-driven inflammatory pathways as potential therapeutic targets. Ongoing work focuses on disseminating these findings through publications and developing translational applications, supported by patents filed during the project.