Atherosclerosis, a major cause of cardiovascular disease [1, 2], involves multiple processes including endothelial cell dysfunction, inflammation, vascular smooth muscle cell (VSMC) proliferation, and matrix alteration.

VSMC proliferation is closely connected to other cellular processes such as inflammation and matrix alterations [3]. More specifically, the switch of VSMCs from contractile type (differentiation) to synthetic type (dedifferentiation) is modulated by growth factors and cytokines secreted during the process of atherosclerosis in vivo. Recent studies showed that the proliferative potential of VSMCs was increased through silencing of geminin [4], which was first identified by Kirschner and colleagues as an inhibitor of DNA replication licensing and a neuralizing factor related to neural cell fate [5, 6]. Geminin, a nuclear protein, is involved in differentiation and cell fate decisions mainly through binding with Cdt1 and Brahma/Brg1 to control the suppression of cellular proliferation and to maintain the undifferentiated state of cells, respectively [7,8,9,10].

Sympathetic nervous activity is also implicated in VSMC proliferation. Neuropeptide Y (NPY), a ubiquitous sympathetic neurotransmitter originally recognized as an appetite stimulant and a vasoconstrictor, recently emerged as a growth factor for a variety of cells including VSMCs. An intriguing observation is that the proliferation-promoting effect of NPY only functions in contractile VSMCs [11]. Moreover, there are multiple receptors, Y1–Y5, mediating the pleiotropic activities of NPY, of which the Y1 receptor is the predominant NPY vascular receptor responsible for accelerating the proliferation of VSMCs. Moreover, norepinephrine coexisting with NPY can also affect VSMC growth by promoting the expression of geminin [12,13,14,15].

With improved understanding of the molecular mechanism of atherosclerosis, the suppression of VSMC growth by targeting cell cycle regulation may be a feasible strategy. In addition, new insights into the role that geminin plays in VSMCs may generate new ideas on treating arteriosclerotic cardiovascular disease in clinical practice.

In this review, we focus on the molecular mechanism of the neurotransmitters of sympathetic postganglionic neurons, especially NPY, that interfere with the proliferation of different VSMCs via regulation of geminin; we also discuss the therapeutic potential of targeting NPY in atherosclerosis from a different perspective.

Molecular function of geminin

In metazoan cells, DNA replication is a complicated process that involves a variety of protein factors. The process must occur once per cell cycle in order to maintain genome integrity. It has been reported that geminin acts as a key regulator of DNA replication licensing through direct binding to Cdt1 and by inhibiting the incorporation of the mini-chromosome maintenance (MCM) complex into the pre-replication complex (pre-RC), thus preventing re-replication in the S phase [8, 16, 17]. The underlying mechanism of cell cycle regulation by geminin is complicated, but there is evidence that a high expression of geminin plays an essential role in suppressing cell proliferation [8]. In addition, geminin can also govern cellular differentiation, not only by regulation of DNA replication, but also through binding of transcription factors such as the Homeobox family [18] and by preventing stem cell differentiation by maintaining the transcription of specific target genes via inhibition of chromatin remodeling activity [7]. Geminin expression determines the fate of a wide variety of cells, for instance, downregulation of geminin protein expression sustains the proliferation potential of hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC; [19,20,21]). Besides, the level of geminin expression in cancer cells is significantly higher than in normal cells in order to prevent the accumulation of stalled replication forks caused by DNA re-replication and avoid apoptosis [22, 23]. Notably, previous studies suggested that there were similar correlations in VSMCs.

Geminin and vascular smooth muscle cells

Zhang and colleagues [4] found that geminin silencing markedly increased the incorporation of 3H-thymidine and EdU in A10, a rat thoracic aortic smooth muscle cell line; this demonstrated that knockdown of geminin can promote DNA synthesis and proliferation of VSMCs. Furthermore, flow cytometry analysis showed that compared with control groups, silencing geminin in VSMCs led to a decrease of G0/G1 cells and an increase of S‑phase cells. Considering that CDK1 is a binding partner of geminin and CDK1 degradation can inhibit replication licensing [24, 25], the researchers investigated whether geminin gene silencing could prevent the degradation of CDK1 and enhance the proliferation potential of VSMCs based on protein and mRNA levels. Collectively, the results of the study indicated that downregulation of geminin facilitated G0/G1–S cell-cycle progression and upregulation of CDK1, which ultimately stimulated the proliferation of VSMCs. In another study, however, the depletion or overexpression of geminin did not significantly affect the proliferation of VSMCs compared with controls [26]. The reason for such diverse results may be due to the different ways of silencing geminin or differences in cell samples.

VSMCs can acquire different functions through phenotypic transformation from a contractile type (differentiation) to a synthetic type (dedifferentiation). VSMC shuttling between the different phenotypes is based on the specific morphology, particularly the varying levels of the marker proteins, and on changes of proliferation potential and migration rates that are higher in synthetic VSMCs than in contractile VSMCs [27, 28]. Interestingly, it has also been reported that geminin, always located in the nucleus, can be excluded from the nucleus to the cytoplasm when contractile VSMCs transformed into synthetic VSMCs, and the same subcellular regulation of geminin was reported in Michigan Cancer Foundation-7 (MCF7) cells as well [29]. Taken together, these data reveal that cell growth and specific cell types are interrelated with the expression levels and subcellular location of geminin, respectively, offering new insight into the shaping of cell destiny by geminin.

Neuronal axis of geminin involved in atherosclerosis

Neurotransmitters of sympathetic postganglionic neurons

As previously noted, increased sympathetic nervous system activity is increasingly regarded as a crucial mechanism underlying cardiovascular complications in humans [30]. In the periphery, elevated levels of circulating norepinephrine, the main neurotransmitter of sympathetic postganglionic neurons, prompt an increase in heart rate, cardiac contractility, vascular tone, and renin–angiotensin system activity [31, 32], making norepinephrine a major contributor to cardiovascular disease. It has been proven that norepinephrine can promote the expression levels of geminin in VSMCs and affect their growth [26]. Interestingly, NPY coexisting with norepinephrine at sympathetic nerve endings in peripheral regions is an endogenous vasoconstrictor and is released with norepinephrine in response to direct stimulation of cardiac sympathetic neurons [12, 13]. On the one hand, in the central nervous system, an increase in NPY in the paraventricular nucleus (PVN) or lateral ventricle can reduce the release of norepinephrine and decrease the activity of peripheral sympathetic nerves, heart rate, and blood pressure. On the other hand, NPY directly binds with the Y1 receptor located on the surface of VSMCs to strengthen the vasoconstrictor effect of norepinephrine. Consequently, sympathetic nervous activity correlates strongly with the pathophysiological process of atherosclerosis via neurotransmitters directly or indirectly affecting vascular endothelial dysfunction and the proliferation of VSMCs.

Geminin and NPY-Y1 receptor system

Besides potentiating norepinephrine activity, NPY significantly enhances the angiotensin II-induced vasoconstrictor response and intensifies the proliferation of VSMCs directly by binding with Y1 receptors to promote atherosclerosis [14, 33]. Moreover, it has been demonstrated that the switch of VSMCs from contractile type (differentiation) to synthetic type (dedifferentiation) plays a critical role in atherosclerosis, and these cell type changes are regulated by cytokines released in atherosclerosis lesions or serum concentrations in vitro [11, 34]. Jiang and colleagues [11] stimulated A10 cells with NPY in different concentrations of serum in order to explore the role of NPY in the proliferation of different VSMC phenotypes in the pathogenesis of atherosclerosis. The results indicated that NPY only promoted the proliferation of VSMCs incubated with 0.5% rather than 10% serum, while VSMCs remained in a differentiated and dedifferentiated state, respectively. Moreover, the authors observed a steep increase of geminin in synthetic-type VSMCs with the presence of NPY, which was attenuated by antagonism of Y1 receptors. Furthermore, geminin prevented DNA replication during the S phase of the cell cycle, and upregulation of geminin induced cell cycle arrest in the S phase. Flow cytometry analysis indicated that more cells remained in the S phase after administration of NPY in synthetic-type VSMCs, which may be mediated by increased expression of geminin.

In the cardiovascular system, harmful vasoconstrictive and proliferative effects of NPY are primarily mediated by Y1 receptors [35]. In in vitro experiments, the proliferative effects of NPY on human pulmonary arterial smooth muscle cells were inhibited in a concentration-dependent way by selective Y1 receptor antagonists [36], while the percentage of cells in the S phase significantly increased via activation of the NPY-Y1 receptor agonist on VSMCs. Thus, this set of results clearly identifies the close connection between VSMC growth induced by NPY and the underlying mechanisms of geminin involvement in atherosclerosis.

Multiple factors released by different cell types such as endothelial cell, platelets, and inflammatory cells induce an aberrant phenotypic switch of VSMCs during atherosclerosis. Additionally, NPY release is long-lastingly activated during the development of atherosclerosis; thus, understanding the relationship between geminin and the NPY-Y1 receptor system may provide exciting insights into treatments for atherosclerosis (Fig. 1).

Fig. 1
figure 1

Effects of the NPY-Y1 receptor system on VSMC proliferation in atherosclerosis via regulation of geminin. In atherosclerosis lesions, multiple regulatory factors are released by different cell types, such as endothelial cell, platelets, and inflammatory cells, which induce an aberrant phenotypic switch of VSMCs from contractile type to synthetic type. Geminin expression in synthetic VSMCs is upregulated by binding of NPY and Y1 receptors that prevents DNA replication during the S phase and leads to cell cycle arrest in the S phase. Thus, stimulation of NPY on VSMCs could be a double-edged sword in the pathogenesis of atherosclerosis. NPY neuropeptide Y, VSMCs vascular smooth muscle cells

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Discussion

Atherosclerosis and its clinical complications such as stroke are common diseases that pose a health threat worldwide. Since Ross proposed that atherosclerosis was a chronic inflammatory disease [37], it has been gradually widely accepted that inflammation as the response to injury represents the pathogenesis of atherosclerosis. On the one hand, the sympathetic nervous system plays a central role in atherosclerosis through release of neurotransmitters to induce inflammation. On the other hand, neurotransmitters such as norepinephrine can interfere with geminin and ultimately affect VSMC proliferation that is linked to atherosclerotic inflammation. Most importantly, the proliferative effects of another neurotransmitter, NPY, on VSMCs can be inhibited by the selective Y1 receptor antagonist [36].

Conclusion

New insights into the neuronal axis of geminin and Y1 receptor antagonist to regulate levels of geminin in VSMCs may provide important knowledge for the treatment of atherosclerosis. This therapy would be aimed at alleviating the side effects of current treatments and lead to a more rational strategy in preventing diseases related to the abnormal transformation of VSMCs.