Pharmacological PDGFRβ inhibitors imatinib and sunitinib cause human brain pericyte death in vitro

King, Natalie E.; Courtney, Jo‐Maree; Brown, Lachlan S.; Foster, Catherine G.; Cashion, Jake M.; Attrill, Emily; Premilovac, Dino; Howells, David W.; Sutherland, Brad A.

doi:10.1016/j.taap.2022.116025

Cited by 9 publications

(18 citation statements)

References 41 publications

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“…To confirm that the proliferative response was mediated by PDGFRβ, HBVP and iPericyte proliferation was assessed in the presence of imatinib. In pericytes, imatinib inhibits PDGFRβ phosphorylation to prevent proliferation (20). In HBVPs and iPericytes, imatinib produced a dose dependent inhibition of PDGF-BB-induced proliferation (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…To expand our knowledge on the relative similarities between mesoderm and neural crest iPericytes, we compared their functional response to PDGF-BB, a growth factor essential for pericyte proliferation and survival (27). We have previously shown that HBVPs proliferate in response to PDGF-BB in vitro through the PDGFRβ receptor (20). Like HBVPs, mesoderm and neural crest iPericytes proliferated in the presence of PDGF-BB.…”

Section: Discussionmentioning

confidence: 99%

“…An EdU assay was used, as described previously (20), to quantify proliferation in mesoderm or neural crest iPericytes compared to HBVPs. Briefly, pericytes were plated in 96 well plates (Interpath, cat.#655180) and grown to 50% confluency (~5,000 cells per well).…”

Section: Proliferation Assaymentioning

confidence: 99%

“…A key ligand-receptor pathway that pericytes utilise for survival and proliferation is the PDGFRβ signalling pathway (20). We exposed HBVPs or iPericytes to basal pericyte medium alone (PM) or PM containing the PDGFRβ ligand, PDGF-BB (100 ng/ml), in the presence of the thymidine analogue, EdU, as previously described (20). The addition of PDGF-BB increased the proportion of HBVPs and iPericytes that incorporated EdU over a 24 h period, indicative of increased proliferation (Fig.…”

Section: Mesoderm and Neural Crest Ipericytes Have Different Gene Exp...mentioning

confidence: 99%

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Induced pluripotent stem cell derived pericytes respond to endogenous mediators of proliferation and contractility

King,

Courtney,

Brown

et al. 2023

Preprint

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BackgroundPericytes are multifunctional contractile cells that reside on capillaries. Pericytes are critical regulators of cerebral blood flow and blood-brain barrier function, and pericyte dysfunction may contribute to the pathophysiology of human neurological diseases including Alzheimers disease, multiple sclerosis, and stroke. Induced pluripotent stem cell (iPSC)-derived pericytes (iPericytes) are a promising tool for vascular research. However, it is unclear how iPericytes functionally compare to primary human brain vascular pericytes (HBVPs). We differentiated iPSCs into iPericytes of either the mesoderm or neural crest lineage using established protocols. We compared iPericyte and HBVP morphologies, quantified gene expression by qPCR and bulk RNA sequencing, and visualised pericyte protein markers by immunocytochemistry. To determine whether the gene expression of neural crest iPericytes, mesoderm iPericytes or HBVPs correlated with their functional characteristicsin vitro, we quantified EdU incorporation following exposure to the key pericyte mitogen, platelet derived growth factor (PDGF)-BB and, contraction and relaxation in response to the vasoconstrictor endothelin-1 or vasodilator adenosine, respectively. iPericytes were morphologically similar to HBVPs and expressed canonical pericyte markers. However, iPericytes had 1864 differentially expressed genes compared to HBVPs, while there were 797 genes differentially expressed between neural crest and mesoderm iPericytes. Consistent with the ability of HBVPs to respond to PDGF-BB signalling, PDGF-BB enhanced and PDGF receptor-beta inhibitors impaired iPericyte proliferation. Administration of endothelin-1 led to iPericyte contraction and adenosine led to iPericyte relaxation, of a magnitude similar to the response evoked in HBVPs. We determined that neural crest iPericytes were less susceptible to PDGFR beta inhibition, but responded most robustly to vasoconstrictive meditators. iPericytes express pericyte-associated genes and proteins and, exhibit an appropriate physiological response upon exposure to a key endogenous mitogen or vasoactive mediators. Therefore, the generation of functional iPericytes would be suitable for use in future investigations exploring pericyte function or dysfunction in neurological diseases.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Proliferation Assaymentioning

confidence: 99%

Section: Mesoderm and Neural Crest Ipericytes Have Different Gene Exp...mentioning

confidence: 99%

See 2 more Smart Citations

Induced pluripotent stem cell derived pericytes respond to endogenous mediators of proliferation and contractility

King,

Courtney,

Brown

et al. 2023

Preprint

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“…Immunocytochemistry for canonical pericyte markers was performed on primary rat pericytes to confirm the purity of cultures. We have previously characterised HBVPs extensively [22][23][24].…”

Section: Immunocytochemistrymentioning

confidence: 99%

Rapamycin Treatment Reduces Brain Pericyte Constriction in Ischemic Stroke

Beard,

Brown,

Morris

et al. 2024

Preprint

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The contraction and subsequent death of brain pericytes may play a role in microvascular no-reflow following the re-opening of an occluded artery during ischemic stroke. Mammalian target of rapamycin (mTOR) inhibition has been shown to reduce motility/contractility of various cancer cell lines and reduce neuronal cell death in stroke. However, the effects of mTOR inhibition on brain pericyte contraction and death during ischemia have not yet been investigated. Cultured pericytes exposed to simulated ischemia for 12 hours in vitro contracted after less than 1 h, which was about 7h prior to cell death. Rapamycin significantly reduced the rate of pericyte contraction during ischemia, however, it did not have a significant effect on pericyte viability at any time point. Rapamycin appeared to reduce pericyte contraction through a RhoA-dependent pathway, independent of changes in intracellular calcium. Using a mouse model of middle cerebral artery occlusion, rapamycin significantly increased the diameter of capillaries underneath pericytes and increased the number of open capillaries 30 minutes following recanalization. Our findings suggest rapamycin may be a useful adjuvant therapeutic to reduce pericyte contraction and improve cerebral reperfusion post-stroke.

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Brain pericytes in culture display diverse morphological and functional phenotypes

et al. 2023

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Pericytes play several important functions in the neurovascular unit including contractile control of capillaries, maintenance of the BBB, regulation of angiogenesis, and neuroinflammation. There exists a continuum of pericyte subtypes along the vascular tree which exhibit both morphological and transcriptomic differences. While different functions have been associated with the pericyte subtypes in vivo, numerous recent publications have used a primary human brain vascular pericytes (HBVP) cell line where this pericyte heterogeneity has not been considered. Here, we used primary HBVP cultures, high-definition imaging, cell motility tracking, and immunocytochemistry to characterise morphology, protein expression, and contractile behaviour to determine whether heterogeneity of pericytes also exists in cultures. We identified five distinct morphological subtypes that were defined using both qualitative criteria and quantitative shape analysis. The proportion of each subtype present within the culture changed as passage number increased, but pericytes did not change morphological subtype over short time periods. The rate and extent of cellular and membrane motility differed across the subtypes. Immunocytochemistry revealed differential expression of alpha-smooth muscle actin (αSMA) across subtypes. αSMA is essential for cell contractility, and consequently, only subtypes with high αSMA expression contracted in response to physiological vasoconstrictors endothelin-1 (ET1) and noradrenaline (NA). We conclude that there are distinct morphological subtypes in HBVP culture, which display different behaviours. This has significance for the use of HBVP when modelling pericyte physiology in vitro where relevance to in vivo pericyte subtypes along the vascular tree must be considered. Graphical abstract

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Pharmacological PDGFRβ inhibitors imatinib and sunitinib cause human brain pericyte death in vitro

Cited by 9 publications

References 41 publications

Induced pluripotent stem cell derived pericytes respond to endogenous mediators of proliferation and contractility

Induced pluripotent stem cell derived pericytes respond to endogenous mediators of proliferation and contractility

Rapamycin Treatment Reduces Brain Pericyte Constriction in Ischemic Stroke

Brain pericytes in culture display diverse morphological and functional phenotypes

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