The global prevalence of cardiovascular diseases (CVD) has led to numerous studies investigating the associated lifestyle-related or genetic risk factors. While the mortality rate of CVD has declined in the past few decades, and improvement in cardiovascular health components was observed at all ages, as the aging population steadily increases worldwide, the projected CVD cost would rise sharply by 2035 (Lloyd-Jones et al., 2010; Virani et al., 2020).

The intactness of endothelium is one of the most fundamental indices of vascular healthiness. Endothelial injury or denudation at the local area as well as a delayed or impaired re-endothelialization process could result in the exposure of the internal membrane and vascular smooth muscle cells (VSMCs) to the circulating blood, creating a pro-thrombotic environment that provoked platelet adhesion and the aggregation of inflammatory cells (Shirali et al., 2016; Mastenbroek et al., 2020). Interestingly, the capacity of endothelial cells (ECs) to restore their integrity from injuries can be enhanced by applying normal shear stress (Xia et al., 2012). The correlations between local wall shear stress (WSS) and atherosclerosis revealed by in vivo observations and computational modeling studies (Heo et al., 2014; Park et al., 2016) also indicate the beneficial roles of steady laminar flow in providing athero-protective effect via regulating EC proliferation and further remodeling process (Gimbrone and Garcia-Cardena, 2013; Roux et al., 2020). On the other hand, disturbed flow found at bifurcations and curvatures posed stress on ECs and resulted in enhanced EC turnover rate at the denuded area (Xu, 2009; Antoniadis et al., 2015).

Lamin A/C, as one of the structural components of the nuclear lamina, not only serves as a mechanically supportive structure but also mediates many cellular events, including cell proliferation by interactions with nuclear membrane proteins and other binding factors (Moiseeva et al., 2011; Vidak et al., 2015). In the vascular system, lamin A/C was also found to be sensitive to mechanical forces and to participate in force-induced cell proliferation, such as its ability to prevent stretch-induced hyperproliferation of VSMC (Han et al., 2015). Genetic mutation in exon 11 on the lamin A/C gene (LMNA) was found to be related to the premature aging process (Simha et al., 2003; Schreiber and Kennedy, 2013). This creates a cryptic splice site responsible for the deletion of 50 amino acids on prelamin A (lamin A precursor) during the post-translational process, resulting in an absence of the recognition site for Zmpste24 (a zinc metallo-endoprotease) to remove the last 15 amino acids at the C-terminus on the precursor. This lack of post-translational modification leads to the generation of a truncated prelamin A called progerin that possesses a permanently farnesylated tail at the C-terminus. Patients carrying that mutation were referred to as having Hutchinson-Gilford progeria syndrome (HGPS). Studies have indicated progerin-induced proliferation impairment in various types of cells and its role in interfering with tissue repair (Wheaton et al., 2017; Hu et al., 2020).

Surprisingly, the McClintock group observed the ubiquitous presence of progerin mRNA transcripts in skin sections derived from healthy donors from all ages, whereas the protein level was elevated with donor’s age (McClintock et al., 2007). Increased misuse of the cryptic splice site on prelamin A was also detected in normal fibroblasts over prolonged in vitro culture regardless of donor’s age. Moreover, mechanisms such as telomere shortening, the hyperactivation of p53 tumor suppressor pathway, the extensive damage in DNA repairing, changes in histone methylation, and other age-dependent factors were also found to play a part in both progerin-induced premature aging and normal aging process (Varela et al., 2005; Benson et al., 2010; Holly et al., 2013; Arancio et al., 2014; Kubben et al., 2016; Kubben and Misteli, 2017). In the vascular system, progerin-induced premature aging syndrome also shared many characteristics with physiological aging at cellular and molecular levels (Olive et al., 2010; Hamczyk et al., 2018a). For example, the Ragnauth group found that lamin A precursor was accumulated in late-passage VSMCs or VSMCs derived from elderly donors, which was proposed to be related to the downregulation of the Zmpste24 gene due to its sensitivity toward oxidative stress over passages (Ragnauth et al., 2010). The evidence suggests that progerin may play a part in vascular aging during the normal aging process.

Considering the low number of HGPS individuals worldwide, many progeroid mouse models have been developed to facilitate HGPS research (Hamczyk et al., 2018a), among which the mice that specifically expressed progerin in the vascular system provided valuable information on how the affected vascular cells contribute to cardiovascular dysfunctions. In these models, restrictive progerin expression in VSMCs resulted in arterial structural alternations and contractile impairment (Del Campo et al., 2019; Del Campo et al., 2020), while endothelial progerin expression tended to provoke systematic inflammatory responses, and the endothelium showed defects in response to shear stress (Osmanagic-Myers et al., 2019; Sun et al., 2020). In a progeroid model where only ECs expressed progerin, premature deaths occurred in these mice at the age approaching 25 weeks, probably due to left ventricle diastolic dysfunction. Extensive interstitial fibrosis and adventitial thickening were also reported through histological examination (Osmanagic-Myers et al., 2019).

Besides extracting cells from progeroid mice models, ECs that expressed progerin were also obtained by differentiation from iPSC (induced pluripotent stem cell) derived from fibroblasts of HGPS patients, or by transfection using constructed progerin plasmids. These progerin-expressing ECs presented features of senescence ECs in terms of shortened telomere length and nuclear dysmorphology, as well as cell cycle arrest and reduced proliferation rate (Matrone et al., 2019; Bidault et al., 2020). Attenuated response of gene regulation toward laminar flow (12 dynes/cm²) was reported, such as atheroprotective gene KLF2 and the downstream targets of NRF2. On the other hand, the expression of pro-inflammatory genes, including E-selection and VCAM-1, was upregulated. Also, observations of altered NOS regulation and abnormal expression levels of elastin and collagen indicated progerin’s impact on endothelial functions in regulating vascular tone (Atchison et al., 2020).

Although many in vitro and in vivo studies have been conducted to examine changes in the phenotypes and gene expression profiles of vascular cells expressing progerin, it is not yet known how EC integrity was compromised by progerin to initiate premature atherosclerosis progression in HGPS patients. On the other hand, while flow pattern has been proven to regulate vascular EC functions, few studies were conducted to reveal the relationship between progerin and the local shear stress environments that ECs may be exposed to. In this study, we established stable cell lines of vascular ECs that overexpressed wild-type lamin A or progerin and studied the effects of progerin on the re-endothelialization process under shear stress. Furthermore, the regenerative capabilities of progeria cells at the wound front under different flow patterns were also examined. Our study helps demonstrate how progerin interferes with endothelial wound repair events in a flow environment.

Recent research has highlighted the importance of endothelial and vascular cell dysfunction in HGPS models, as well as progerin-induced mechano-sensitivity in the endothelium (Benedicto et al., 2021). While progerin in endothelial cells has been shown to induce inflammatory responses and impaired mechanotransduction (Osmanagic-Myers et al., 2019). this is a more specific study on how progerin interferes with an endothelial wound recovery. Vascular endothelial injury that may occur in vivo following endothelial apoptosis or during post-angioplasty restenosis disrupts endothelial integrity and permeability. Endothelial denudation would expose other vascular cells to the blood flow and trigger a cascade of inflammatory responses (Durand et al., 2004; Curcio et al., 2011). The subsequent healing responses involve endothelial proliferation and migration that occurs under blood flow in vivo. Recent studies have revealed the key role of progerin in vascular aging as well as the presence of similar aberrant lamin A isoform in aged people (Minamino and Komuro, 2008; Olive et al., 2010; Ragnauth et al., 2010; Gerhard-Herman et al., 2012). Progerin was also found to impair VSMC proliferation and the metabolism of low-density lipoprotein that involved crosstalk between ECs and VSMCs (Hamczyk et al., 2018b; Goldberg et al., 2018; Hamczyk et al., 2019).

In this paper, we were focusing on the effect that progerin has on endothelial healing response upon denudation under flow, as well as the how cell division and cell cycle during wound recovery are affected in progerin-expressing ECs. We hypothesize that progerin interferes with endothelial integrity under physiological flow and provided evidence of progerin-induced endothelial wound healing delay under normal shear stress (Figure 4). Furthermore, we investigated cell proliferation activities at wound front and wound healing direction relative to flow and found that late mitosis cells at wound edges downstream of flow were most affected by shear stress during the repair event (Figure 6).

Stable cell lines that expressed either wild-type lamin A or progerin protein were generated using a retrovirus packaging system. Wild-type lamin A-expressing cells exhibited nuclei foci that could be due to the overexpression of the external protein, and progerin-expressing cells were characterized as more wrinkles and folds on nuclei. Subsequent wound healing assay revealed delay in endothelial repair in progerin-expressing cells with the application of physiological shear stress, regardless of wound directions (Figure 4). Further data analysis indicated that cell proliferation abilities were compromised at wound front in these ECs, where the number of cells at late mitosis was significantly reduced at vertical wound edges downstream of flow at normal shear stress compared to low shear (Figure 6). These data suggest that endothelial recovery in progerin-expressing ECs was particularly sensitive to physiological shear when it is against the direction of wound recovery.

Additionally, progerin-expressing cells also showed reduced fraction of cells undergoing DNA replication under flow, although the DNA synthesis rate was comparable between wild-type lamin A- and progerin-expressing cells under static state (Figure 5). Normal shear stress reduced number of cells in S phase in WT lamin A-expressing cells, which agrees with previous studies that showed laminar physiological suppressed proliferation as a way to maintain EC quiescent phenotype at athero-protective regions (Chistiakov et al., 2017). The attenuation of fraction of cells in S phase by normal shear is not seen in progerin-expressing cells, which suggests their loss of mechano-sensitivity to shear stress, in regulating the cell cycle.

On the other hand, the number of binucleated cells at the wound front is more prominent in progerin ECs under different patterns of flow (Figure 6). In normal tissues and tumors, cells with double nuclei were also observed and are thought be a result of cytokinesis failure or cell fusion (Nishimura et al., 2016). Although binucleated cells were also present in healthy primary culture, the amount was dramatically increased in HGPS fibroblast and adipocytes, which could be related to abnormal chromosome segregation and accelerated cell senescence (Dominici et al., 2001; Cao et al., 2007; Xiong et al., 2013). However, the underlying mechanisms of binucleated cells remain unknown, and the clinical significance is controversial (Chen et al., 2007).

In summary, we found decreased wound healing rate in progerin-expressing ECs under physiological level of laminar shear stress compared with those under low magnitude shear, and the effect was independent on wound orientations relative to flow direction. Further examination showed that the delayed wound recovery coincided with a reduced number of cells at late mitosis, suggesting potential interference by progerin with delayed cell division. Attenuation of DNA synthesis by normal level of shear stress observed in WT lamin A endothelial cells also disappeared in progerin cells. Both findings support the conclusion that endothelial responses toward athero-protective levels of shear stress were not seen in progerin-expressing ECs, which demonstrates a reduced sensitivity toward mechanical stress in endothelial cells in the presence of progerin. This finding is also in agreement with other recent studies on progerin-expressing endothelium (Osmanagic-Myers et al., 2019).

Research on how progerin affects vascular aging is still ongoing. For example, the exact isoform of prelamin A in elderly people remains unclear. The detection of progerin in cells or tissues derived from normal healthy people has been reported (Scaffidi and Misteli, 2006; McClintock et al., 2007), while the accumulation of the farnesylated prelamin A that retains the 50 amino acids near the carboxyl-terminus was observed in late-passage VSMCs or those from aged donors (Ragnauth et al., 2010). Although the difference in those prelamin A isoforms seemed to be minor in structure, its accumulation mechanisms and downstream effects could be varied.

Moreover, it is still unclear if the toxicity of progerin in HGPS patients was induced by its farnesyl lipid anchor or the 50-amino-acid deletion near the C-terminus. This stems from the purpose of prelamin A processing based on our current understanding. It was found that prelamin A farnesylation in mammalian cells was critical in anchoring lamin A to nuclear lamina during interphase (Lutz et al., 1992; Hennekes and Nigg, 1994). The absence of the farnesylation step on prelamin A also resulted in reduced binding affinity with heterochromatin in HEK293 cells (Lattanzi et al., 2007). However, further studies revealed that the elimination of the farnesylated tail on progerin did not rescue all phenotypes in HGPS cells (Verstraeten et al., 2008). A mouse model that bypassed the modifications and only expressed mature lamin A exhibited normal body weight and survived without developing severe disease phenotypes (Yang et al., 2008; Coffinier et al., 2010).

Current treatment plan for HGPS is still being under development. The first and only drug approved by the US Food and Drug Administration (FDA), Zokinvy (lonafarnib), is a farnesyltransferase inhibitor that detaches progerin from the nuclear membrane. However, farnesyltransferase inhibitors did not rescue all phenotypes in HGPS cells, probably due to the retainment of progerin within nucleoplasm (Verstraeten et al., 2008). Other therapies have been recognized recently, such as isoprenylcysteine carboxyl methyltransferase (ICMT) inhibitor and progerin-lamin A binding inhibitors (Chen et al., 2021; Kang et al., 2021; Macicior et al., 2021; Marcos-Ramiro et al., 2021). More investigations in the functional roles of the deleted 50 amino acids could shed light on alleviating the effect of progerin as well as better HGPS treatment regimens (Lai and Wong, 2020).

We are currently working to continue to examine the mechanistic effect of progerin in shear-induced endothelial functions such as migration, which also plays a key role in the re-endothelialization process. Our findings provided insights into how progerin affects endothelial mechanotransduction in our effort to understand the presence of plaques in HGPS vasculature (Hamczyk et al., 2018a); while further emphasizing the importance of physiological shear in promoting and maintaining endothelial integrity.