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. 2022 Mar 21:13:867070.
doi: 10.3389/fphar.2022.867070. eCollection 2022.

Pericytes' Circadian Clock Affects Endothelial Cells' Synchronization and Angiogenesis in a 3D Tissue Engineered Scaffold

Valeria Mastrullo  1   2   3 , Daan R van der Veen  2 , Priyanka Gupta  3 , Rolando S Matos  1 , Jonathan D Johnston  2 , John H McVey  1 , Paolo Madeddu  4 , Eirini G Velliou  3   5 , Paola Campagnolo  1
Affiliations

Pericytes' Circadian Clock Affects Endothelial Cells' Synchronization and Angiogenesis in a 3D Tissue Engineered Scaffold

Valeria Mastrullo et al. Front Pharmacol. .

Abstract

Angiogenesis, the formation of new capillaries from existing ones, is a fundamental process in regenerative medicine and tissue engineering. While it is known to be affected by circadian rhythms in vivo, its peripheral regulation within the vasculature and the role it performs in regulating the interplay between vascular cells have not yet been investigated. Peripheral clocks within the vasculature have been described in the endothelium and in smooth muscle cells. However, to date, scarce evidence has been presented regarding pericytes, a perivascular cell population deeply involved in the regulation of angiogenesis and vessel maturation, as well as endothelial function and homeostasis. More crucially, pericytes are also a promising source of cells for cell therapy and tissue engineering. Here, we established that human primary pericytes express key circadian genes and proteins in a rhythmic fashion upon synchronization. Conversely, we did not detect the same patterns in cultured endothelial cells. In line with these results, pericytes' viability was disproportionately affected by circadian cycle disruption, as compared to endothelial cells. Interestingly, endothelial cells' rhythm could be induced following exposure to synchronized pericytes in a contact co-culture. We propose that this mechanism could be linked to the altered release/uptake pattern of lactate, a known mediator of cell-cell interaction which was specifically altered in pericytes by the knockout of the key circadian regulator Bmal1. In an angiogenesis assay, the maturation of vessel-like structures was affected only when both endothelial cells and pericytes did not express Bmal1, indicating a compensation system. In a 3D tissue engineering scaffold, a synchronized clock supported a more structured organization of cells around the scaffold pores, and a maturation of vascular structures. Our results demonstrate that pericytes play a critical role in regulating the circadian rhythms in endothelial cells, and that silencing this system disproportionately affects their pro-angiogenic function. Particularly, in the context of tissue engineering and regenerative medicine, considering the effect of circadian rhythms may be critical for the development of mature vascular structures and to obtain the maximal reparative effect.

Keywords: angiogenesis; circadian; pericytes; tissue engineering and regenerative medicine; vasculature.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Saphenous vein derived pericytes (SVP) possess an endogenous molecular clock. mRNA expression of Per2 (N = 3, (A)), Bmal1 (N = 3, (B)) and Rev-erbα (N = 3, (C)), in SVP cells. Data is presented as 2−ΔΔCt and normalized on β-actin housekeeping mRNA expression. Error bars represent SEM and 95% CI is shown in the graph. Non-linear regression (Cosinor Curve) is over-imposed and favorably compared against straight line. Radar graph denoting the peak of mRNA expression of each gene over 24 h (D). Immunofluorescence representative images (T16 and T32) and integrated density quantification of nuclear signal intensity of proteins BMAL1 (N = 3, (E)) and REV-ERBα (N = 3, (F)). Error bars represent SEM and 95% CI are shown in the graph. Non-linear regression (Cosinor Curve, fixed period of 24 h) is over-imposed and favorably compared against straight line (p < 0.05). DAPI blue, Phalloidin red, BMAL11/REV-ERBα green, scale bar: 100 µm. Average detrended oscillatory profile of serum shock synchronized SVP cells transduced with Per2:Luc (N = 2, (G)) Bmal1:Luc (N = 2, (H)) or Rev-erbα:Luc (N = 2, (I)) lentivectors. Red line represents a damped sin wave with a period of 24 h.
FIGURE 2
FIGURE 2
Pericytes’ clock synchronization influences endothelial cells’ Bmal1 rhythmicity. mRNA expression of Bmal1 (N = 3, (A)) and Rev-erbα (N = 3, (B)) in human umbilical vein endothelial cells (HUVEC). The data are shown as 2−ΔΔCt and normalized on β-actin housekeeping mRNA expression. Error bars represent SEM and 95% CI is shown in the graph. Non-linear regression (Cosinor Curve) was compared against straight line; straight line is the preferred model (p > 0.05). Average detrended non-oscillatory profile of serum shock synchronized HUVEC cells transduced with Bmal1:Luc (N = 2, (C)) or Rev-erbα:Luc (N = 2, (D)) lentivectors. Data are shown in area graphs and counts/sec are plotted against days post-synchronization. Red line represents a damped sin wave with a period of 24 h. Average detrended oscillatory profile of Bmal1:Luc in HUVEC cells in a contact (N = 3, (E)) and non-contact (N = 3, (F)) co-culture with synchronized saphenous vein pericytes (SVP) Image shows a schematic of the protocol. Red line represents a damped sin wave with a period of 24 h.
FIGURE 3
FIGURE 3
Clock disruption affects tube formation in Matrigel assay. Bmal1 mRNA expression in saphenous vein pericytes (SVP, (A)) and in human umbilical vein endothelial cells (HUVEC, (C)) in knockdown cells (shBMAL1) in comparison with control cells (shNEG). The data are shown as 2−ΔΔCt and normalized on β-actin housekeeping mRNA expression. Fold change in viability and apoptosis of shBMAL1 cells over time, relative to shNEG cells in SVP (n = 4, (B)) and HUVEC (n = 4, (D)), respectively. Relative average branch thickness ((E), Diameter), network coverage ((F), Integrated density) and total branch length ((G), Total length) measured in HUVEC (H) cultured alone or in co-culture with SVP (S) on Matrigel. Different combination of shNEG cells (N) and shBMAL1 (B) cells are compared. Representative mask pictures of the analysis are shown. Data were analyzed using two-way ANOVA, and * = p < 0.05, ** = p < 0.01, *** = p < 0.001 vs. shNEG or wild-type co-cultures.
FIGURE 4
FIGURE 4
Pericytes’ clock influences endothelial cells’ lactate release. Lactate accumulation in the supernatants of saphenous vein pericytes (SVP, S), transduced with control lentivirus (shNEG) or shBMAL1, and either co-cultured with wild-type human umbilical endothelial cells (HUVEC, H) (A) or cultured alone (B). Linear regression is over-imposed. Incremental change (Δ) in lactate and glucose concentration in the supernatants of SVP shNEG (C) and SVP shBMAL1 (D), relative to previous timepoint.
FIGURE 5
FIGURE 5
Distribution of endothelial cells and pericytes in a 3D polyurethane scaffold. Representative immunofluorescence images of scaffolds seeded with human umbilical vein endothelial cells (HUVEC, (A)), saphenous vein pericytes (SVP, (B)) and a co-culture of HUVEC and SVP (C). CD31 (red), NG2 (green) and DAPI (blue). Scale bar:100 µm. Quantification of relative cell distribution in the scaffold, in synchronized (synch, (D)) and non-synchronized (non-synch, (E)) co-cultures. Heat maps show the relative frequency up to 750 pixels of distance. Data were analyzed using two-way ANOVA, and **** = p < 0.0001.
FIGURE 6
FIGURE 6
Clock synchronization improves vascular structure formation in 3D. Representative immunofluorescence images of synchronized (Synch) and non-synchronized (Non-synch) co-cultures on poly-urethane scaffolds, at different time-points [(A), N = 3 for each time point]. Endothelial cells (CD31, red) and pericytes (NG2, green), nuclei in blue (DAPI). Scale bars: 100 µm Quantification of the number of endothelial cells (B) and pericytes (C) relative to the total number of cells in each pore, total number of cells per pore (D) and percentage of pore coverage (E) was compared in Synch and Non-synch conditions. Two-way ANOVA, * = p < 0.05, ** = p < 0.01 vs. Non-synch at each time-point.
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