Mitochondrial transfer from pericytes to endothelial cells – a central switch between maintenance of vascular homeostasis and pathological vascular remodeling

Principal Investigator

Scientific interest within the context of the graduate college

Pulmonary hypertension is a common cardiopulmonary disease that is characterized by extensive remodeling of the pulmonary vascular tree and a poor prognosis due to ultimate right heart failure. The underlying progressive lung vascular remodeling involves two distinct but interconnected pathologies: distal capillary loss (vascular rarefaction) and proliferative remodeling of precapillary resistance vessels. Current therapeutic approaches rely on vasodilatory drugs and do not address the underlying causes of vascular remodeling which are poorly understood. Yet, deeper insight into the underlying pathophysiological processes – which forms the basis for the development of causal rather than symptomatic therapies – is hampered by limited access to human biosamples, the restriction to post-mortem end-point analyses in animal models, and the complexity of the multicellular processes driving vascular remodeling which cannot be recapitulated in traditional cell culture. To overcome this gap in methodologies, knowledge and ultimately therapy, we have engaged in a collaboration with a Suisse bioengineering lab to develop in-vitro microvasculature-on-chip and artery-on-chip models that allow for the first time to track changes of the vascular system and the dynamics of individual cell types in an unprecedented temporally and spatially resolved context.¹ These models – in combination with advanced imaging modalities, state-of-the-art metabolic assays and functional read-outs – are now ready to use and open up unprecedented avenues for the discovery of new mechanisms of health and disease and the development of new therapeutic targets.

Project description

Pulmonary hypertension is an ultimately fatal disease characterized by an increase in pulmonary vascular resistance that results from a combination of capillary rarefaction and inward remodeling of pulmonary arteri(ol)es.² Recently, pericytes have been implicated in both of these events: Pericytes are perivascular cells that are encased within the microvascular basement membrane and assist in the maturation and stabilization of microvascular networks. To this end, pericytes closely communicate with endothelial cells promoting the stabilization of endothelial cells and the maintenance of vascular barrier function. In vitro, pericytes can even “donate”, i.e. transfer mitochondria to endothelial cells via so-called tunnelling nanotubes (TNTs).³

In pulmonary hypertension, however, pericytes detach from capillary networks, possibly contributing to their disintegration.⁴ In parallel, pericytes emerge at the level of pulmonary arteries where they integrate into the muscular layer of the vascular wall likely contributing to medial thickening.⁵ In previous work, we have shown i) that pericytes form TNTs with lung microvascular endothelial cells in intact lung capillary networks, ii) that these interaction sites show abundant expression of connexin 43, a gap junctional molecule known to coordinate mitochondrial transfer between cells,⁶ and iii) that pulmonary artery endothelial cells in pulmonary hypertension undergo a metabolic switch from oxidative phosphorylation to glycolysis (a phenomenon known from cancer cells as “Warburg-effect” that allows cancer cells to hyperproliferate). Based on these observations and published data demonstrating a similar metabolic switch in pericytes in pulmonary hypertension,⁷ we propose a paradigm-shifting metabolic concept for the co-occurrence of capillary loss and arteriolar remodeling in pulmonary hypertension that positions the pericyte in the center of the pathophysiology: In healthy homeostasis, pericytes closely interact with microvascular endothelial cells, supporting their oxidative phosphorylation by transferring mitochondria in a connexing 43-dependent manner to sustain capillary homeostasis. In pulmonary hypertension, pericytes undergo metabolic reprogramming toward increased glycolysis, resulting in their detachment from capillaries and migration to upstream resistance vessels where they integrate into the vascular wall, transferring glycolytic mitochondria to pulmonary artery smooth muscle cells thus in turn promoting their hyperproliferation and subsequent arteriolar remodeling while capillaries now disassemble in the absence of mitochondrial support from pericytes.

Our microvasculature-on-chip and artery-on-chip models in conjunction with classic co-culture systems are uniquely positioned to address this hypothesis via the following research aims:

Aim 1. To map mitochondrial transfer dynamics in lung capillary networks in health and pulmonary hypertension, we will use multi-color confocal and multi-photon imaging to track in real-time the interaction between pericytes and endothelial cells (identified by different fluorescent markers) and the transfer of fluorescently labelled mitochondria from one to another via TNTs. Experiments will be run in vascular networks formed by primary vascular cells from healthy donors and those from patients with pulmonary hypertension with the expectation that in the latter scenario, pericytes will detach from the capillaries and stop transferring mitochondria to lung microvascular endothelial cells, resulting in capillary disintegration and network rarefaction. Using dedicated assays for mitochondrial metabolism (Seahorse stress assays, Oroboros assays, metabolomic analyses by GC-MS) we will analyze the effects of this mitochondrial transfer (or lack thereof) on endothelial metabolism and function (in terms of proliferation versus apoptosis). The proposed regulatory role of connexin 43 will be tested using pharmacological inhibitors or enhancers of connexin 43-mediated gap junctional communication.

Aim 2. Using a newly engineered arteriole-on-a-chip platform, we will next probe whether pericytes with a pharmacologically or genetically induced glycolytic shift preferentially integrate into the arterial wall and (instead of promoting oxidative phosphorylation in lung capillaries) now start to donate glycolytic mitochondria to pulmonary artery smooth muscle cells, driving their proliferation. An analogous set of imaging, metabol(om)ic and functional assays will be applied as for Aim 1.

Aim 3. Finally, we will utilize a presently custom-designed platform combining a capillary network with a feeding arteriole on a single organ-on-a-chip to track the complete cascade of events from initial capillary homeostasis with quiescent pericytes to pericyte glycolytic switching, detachment and migration to the upstream arteriole where they ultimately trigger vascular remodeling by donating “injurious”, i.e. glycolytic mitochondria to smooth muscle cells.

The results of the proposed are expected to radically reshape our understanding of pulmonary hypertension, positioning changes in pericyte metabolism at the very center of the pathophysiology and identifying strategies to restore pericyte quiescence and homeostatic metabolism as a promising new avenue for the treatment of a presently uncurable disease.

Application details

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