CE14 - Physiologie et physiopathologie

To capitalize on activated adventitial fibroblasts metabolism to treat pulmonary hypertension – Gly-Pro-PH

To capitalize on activated adventitial fibroblasts metabolism to treat pulmonary hypertension

Pulmonary hypertension (PH) is a deadly enigmatic disease of the pulmonary vasculature that is becoming increasingly prevalent worldwide with substantial unmet medical needs. In response to hormonal, inflammatory and environmental stresses such as hypoxia/ischemia, or vascular distention, pulmonary artery adventitial fibroblasts (PAAF) are the first vascular wall cells to exhibit evidence of activation.

To delineate the metabolic landscape of PAAFs and its regulatory network to guide the development of novel therapeutic strategies centred on PAAF metabolism.

Vascular stiffness and extracellular matrix (ECM) remodelling promote pulmonary vascular dysfunction early in PH pathogenesis. Although anatomic location may dictate distinct effects on disease progression, stiffness in both the proximal and distal pulmonary arterial tree is important for overall pathogenesis. Importantly, abnormal collagen and elastin deposition and extracellular matrix remodelling worsen stiffness and affect both proximal and distal pulmonary vessels in PH. Furthermore, stiffness is associated with the degree of right ventricle dysfunction, dilation and hypertrophy in PH. The structural alterations in the pulmonary vasculature are arbitrated by activated vascular cells, which acquired and exhibited hyperproliferative, migratory and invasive capabilities as well as metabolic reprogramming. In response to hormonal, inflammatory and environmental stresses such as hypoxia/ischemia, or vascular distention, resident PAAFs are the first vascular wall cells to exhibit evidence of “activation”. Such activation is characterized by increases in cellular proliferation, in the expression of contractile and ECM proteins, as well as in the secretion of chemokines, cytokines, growth, and angiogenic factors capable of directly affecting resident vascular wall cell growth and of initiating inflammation in a manner that influences overall vascular tone and wall structure. Classical vascular ECM remodelling occurs through changes in the balance between collagen and elastin deposition, matrix degradation, and collagen crosslinking enzymes such as lysyl oxidase (LOX) -- all processes that are energy requiring. Recently, studies have proposed that this phenotype is partly explained by reprogramming of arterial and surrounding stromal cell metabolic processes. However, beyond glycolysis, the metabolic requirement of activated PAAFs remain unknown. Moreover, the timing of such changes in the course of disease has not been established, and only recently have such technologies been developed to track these molecular changes in relation to PAH progression. Yet, deciphering the metabolic circuits of PAAF activation will offer new insight into the molecular underpinnings of this disease and provide novel therapeutic entry points for intervention strategies.

A better characterization of the causative relationship between activated fibroblast-dependent ECM remodelling, its biochemistry and organization, and cell metabolism is needed. To progress with this goal, we propose to leverage from the recognized expertise of our consortium in cell metabolism, vascular cell biology, fibroblast activation, ECM remodelling and PH. We will combine in vitro models allowing studies in controlled environment with in vivo rodent models and patient tissues, allowing to establish the physio-pathological relevance of our findings. We will leverage the internationally recognized expertise of the technical platforms of our institutes/departments (i.e. metabolomics, proteomics, functional exploration, microscopy, Atomic Force Microscopy (AFM), among other) to effectively link vascular cells activation and metabolic rewiring of vascular cells as two integrally related molecular drivers of ECM remodelling and stiffening during PH evolution.

Thanks to the transplantation activity in Marie Lannelongue hospital linked to the French Referral Centre for Pulmonary Hypertension (Kremlin-Bicêtre hospital) (Partner 1 – U999), we have obtained all primary cultures of PAAF from PAH and control (non-PAH) patients needed for this project. Using those cells, partner 2 (UMR-7275) found that PAAF activation by either stiffness, inflammatory cytokine (IL-6, IL-1alpha, TGF-beta), or hypoxia converges on the rewiring of proline and glycine metabolism. Together, our results indicate that collagen secretion, responsible for perivascular pulmonary remodeling and stiffening, is concomitant to PAAF activation and that PAAF activation rewires the proline/glycine metabolism.
Although activated PAAF proline biosynthesis relies mainly on glutamine (Gln) catabolism, our results indicate that other carbon sources could bypass Gln restriction, such as glutamate (Glu), serine (Ser), or proline (Pro) itself. Therefore, we investigated the effect of the Glu Pro Gly Ser Deficient diet on proline synthesis, perivascular collagen deposition, pulmonary arteriolar stiffening, and subsequent hemodynamic effects on the rat model of monocrotaline (MCT)-induced PAH. MCT-exposed rats fed with a non-deficient diet developed severe PAH characterized by high pulmonary artery pressure (PAP) and right ventricle hypertrophy due to occlusive distal pulmonary vascular remodeling. These effects were significantly attenuated with the Glu Pro Gly Ser Deficient diet. Restriction diet has recently gained traction to prevent and or cure diseases. The results of our Gly-Pro-PH ANR project provide novel and not previously considered therapeutic strategies centered on activated-PAAF metabolism via Glu Pro Gly Ser restriction.

siRNA knockdown or pharmacological inhibition of either GLS (the enzyme that converts the glutamine to glutamate, the first step of the glutamine to proline metabolic pathway) or SHMT1 (the enzyme that converts the serine to glycine) impacted the Pro and Ser metabolism, decreased collagen production and ECM remodeling in IL6-activated fibroblasts and PAAFs isolated from PH patients. The next step will be to test SHMT1 and GLS inhibitors in various models of PH.

1. Torrino S et al. Metabo-reciprocity in cell mechanics: feeling the demands/feeding the demand. Trends Cell Biol. 2022 Feb 14:S0962-8924(22)00028-9. doi: 10.1016/j.tcb.2022.01.013.

2. Gallerand A, et al. Brown adipose tissue monocytes support tissue expansion. Nat Commun. 2021 Sep 6;12(1):5255.

3. Torrino S, et al. Mechano-induced cell metabolism promotes microtubule glutamylation to force metastasis. Cell Metab. 2021 Jul 6;33(7):1342-1357.e10.

4. Acharya AP, et al. Simultaneous Pharmacologic Inhibition of Yes-Associated Protein 1 and Glutaminase 1 via Inhaled Poly(Lactic-co-Glycolic) Acid-Encapsulated Microparticles Improves Pulmonary Hypertension. J Am Heart Assoc. 2021 Jun 15;10(12):e019091.

Pulmonary arterial hypertension (PAH) is a deadly enigmatic disease of the pulmonary vasculature with substantial unmet medical needs. PAH is characterized by a complex panvasculopathy involving excessive proliferation and dysregulation of multiple cell types, as well as inflammation and fibrosis throughout the vasculature, leading to increase pulmonary arterial pressure and ultimately right heart failure. PAH can be idiopathic or heritable mainly due to mutations in the BMPR2 gene. Recently, we and other have demonstrated that dysregulated vascular cell metabolism and perivascular extracellular matrix (ECM) stiffening triggers PAH development. In response to multiple PAH triggers such as, hormonal, inflammatory and environmental stresses (hypoxia/ischemia), or vascular distention, pulmonary artery adventitial fibroblasts (PAAF) are the first vascular wall cells to exhibit evidence of activation. Activated PAAFs promote perivascular collagen deposition and actively remodelled ECM --all processes that are energy requiring. Yet, it remains unknown whether and how vascular cell metabolism rewiring supports the metabolic needs of activated PAAF and affect ECM remodelling.

Manifestations of symptoms including shortness of breath and right heart failure often come late in the course of the disease. Therefore, there is a short delay between diagnosis and outcome, and relevant pulmonary samples in PAH can only be obtained either at end-stage disease upon patient death or after lung transplantation. This inherently limits our ability to obtain a phenotype/molecular disease signature during critical time points, such as the disease initiation and its development over a temporal framework. To gain insight in the molecular underpinnings of PAH, animal models have been developed. Despite extensive endeavours no animal model adequately represents human PAH and often do not allow study of the disease development time course. To capture the molecular underpinning of PAH disease we developed the first Bmpr2 mutant rats. Strikingly, this rat recapitulates critical cellular and molecular dysfunctions described in human PAH. Perhaps most importantly this rat develops a gradated and time-dependent PAH, thus allowing molecular and cellular studies to define the origin of PAH evolution.

Therefore, we propose to leverage on a unique combination of primary cells isolated from PAH patients, rodent models of PAH, and human biopsies from a large biobank of PAH plasma and tissues to decipher the causative relationship between PAAF activation, PAAF metabolism and perivascular ECM remodelling and stiffening during PAH evolution. Coupled to a combination of transdisciplinary approaches including molecular biology, metabolomics, system-levels biology and preclinical models we propose to unveil the molecular underpinning of ECM remodelling and stiffening in PAH.

Ultimately, deciphering the causative relationship between activated PAAF metabolism and collagen synthesis, as well as reveal the molecular circuit that control the metabolic rewiring of PAAF, will provide critical insight in the molecular origin of PAH, thus providing novel and not previously considered therapeutic strategies centred on PAAF metabolism.

Project coordination

Frédéric PERROS (Hypertension artérielle, pulmonaire : physiopathologie et innovation thérapeutique)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

IPMC Institut de pharmacologie moléculaire et cellulaire
U999 Hypertension artérielle, pulmonaire : physiopathologie et innovation thérapeutique

Help of the ANR 603,560 euros
Beginning and duration of the scientific project: December 2020 - 48 Months

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