Double-Face of Vasohibin-1 for the Maintenance of Vascular Homeostasis and Healthy Longevity

Abstract

The structural and functional integrity of endothelium is essential for the maintenance of vascular health. Vasohibin-1 (VASH1), originally isolated as an endothelium-derived angiogenesis inhibitor, has another function to promote stress tolerance of endothelial cells (ECs), and these functions are critical for the maintenance of vascular homeostasis preventing both pathological angiogenesis and stress-induced vascular diseases. The expression of VASH1 is downregulated during replicative senescence of ECs by the alteration of microRNA expression, and this age-associated downregulation of VASH1 might be a risk of deterioration of vascular homeostasis and age-related vascular diseases. Contrary to this expectation, the lack of Vash1 gene in mice exhibited healthy longevity. Thus, VASH1 has double-face for the maintenance of vascular homeostasis and healthy longevity. This feature of VASH1 and its mechanism will be described in this mini review.

Introduction

Cardiovascular diseases (CVDs) are the major cause of death in industrialized countries. Aging is the critical risk factor of CVDs, as the prevalence of CVDs is significantly increased in the older population¹⁾. Aging is associated with the progressive deterioration of the structure and function of all cell types, including vascular endothelial cells (ECs). Aging is influenced both by replicative senescence and by environmental factors. When various cellular stresses cause DNA damage, cells arrest their cell cycle for the repair of DNA damage, which leads to premature cell senescence. Replicative senescence is the result of telomere shortening, whereas premature cell senescence does not require such an event. Nonetheless, replicative senescent cells are more prone to DNA damage and the resultant premature cell senescence²⁾.

ECs on the vascular wall receive cumulative damages during aging, and this causes the age-dependent impairment of the vascular endothelium³⁾. In order to maintain the integrity of vascular structure and function, the vascular endothelium possess a self-defense system. Vasohibin-1 (VASH1) is initially isolated as an endothelium-derived angiogenesis inhibitor, and is further revealed to promote stress tolerance of ECs for the maintenance of vascular homeostasis^{4, 5)}.

Isolation of Vasohibins

To search for angiogenesis regulators expressed in ECs, we stimulated ECs in culture with VEGF and then performed DNA microarray analysis to characterize VEGF-inducible genes in these cells. We focused our attention on genes whose functions were undefined at that time. Recombinant proteins of such genes were made and applied to functional bioassays of angiogenesis. This attempt led to the isolation of a gene having anti-angiogenic activity, which we designated as vasohibin (VASH). By examining in a database, we found a gene homologous to VASH, and designated the homolog as vasohibin-2 (VASH2) and we rename VASH as VASH1. The overall homology between human VASH1 and human VASH2 is 52.5% at the level of the amino-acid sequence. Moreover, it is now revealed that lower organisms possess one ancestral vasohibin gene, whereas vertebrates have VASH1 and VASH2. The common ancestral vasohibin gene is divided into VASH1 gene and VASH2 gene during the evolution. The conserved amino-acid sequence across species indicates the importance of the biological role of the vasohibin family⁶⁾.

VASH1 for Angiogenesis Inhibition and Stress Tolerance of ECs

VASH1 is preferentially expressed in ECs of newly formed blood vessels behind the sprouting front where angiogenesis terminates⁷⁾. When angiogenesis was induced in Vash1 KO mice, angiogenesis was maintained in the area where angiogenesis should be terminated. Moreover, when the adenoviral vector encoding human VASH1 (AdhVASH1) was injected from tail vein of Vash1 KO mice, this sustained angiogenesis was abrogated⁷⁾.

Besides its emergence there on the vascular wall for the termination of angiogenesis, the VASH1 protein is immunohistochemically detectable in certain ECs unrelated to angiogenesis⁸⁾. The additional role of VASH1 was subsequently revealed by the knockdown of VASH1 in cultured HUVECs, which treatment significantly increased the number of premature senescent cells. Alternatively, when VASH1 was overexpressed in HUVECs, such cells became resistant to stress-induced premature cell senescence⁵⁾. This protective function of VASH1 was confirmed under in vivo conditions by the treatment of mice with paraquat, which generates superoxide and causes acute organ damage including lungs. When compared with wild-type mice, Vash1 KO mice died in greater numbers due to acute lung injury caused by paraquat. Alternatively, when VASH1 was overexpressed in the lungs of Vash1 KO mice by the intra-tracheal administration of Adh-VASH1, the paraquat-induced acute lung injury was almost completely prevented⁵⁾. The protective role of VASH1 from vascular diseases was further clarified by comparing the development of STZ-induced diabetic nephropathy, or the development of intimal thickening of femoral artery in cuff-injury model in Vash1 KO mice with that in their wild-type mice. The results indicated that diabetic nephropathy or intimal thickening of femoral artery was significantly exacerbated in Vash1 KO mice^{9, 10)}. The protective activity of VASH1 was further documented in murine diabetes models, as human VASH1 protein delivered from the liver infected with AdhVASH1 prevented diabetic nephropathy^11,12) or intimal thickening of femoral artery¹³⁾.

Thus, these loss-of-function and gain-of-function studies have clearly demonstrated the role of VASH1 is to maintain vascular homeostasis by both angiogenesis inhibition and stress tolerance of ECs.

Dual Regulation of VASH1 Expression for the Maintenance of Vascular Homeostasis

Gene expression begins with the transcription of DNA for the generation of mRNA and ends with its translation to protein. When ECs are exposed to pro-angiogenic stimuli such as VEGF, ECs increase their expression of VASH1 mRNA and VASH1 protein synthesis over a 24-hour period⁴⁾. This VEGF-mediated increase of VASH1 mRNA is not influenced by the actinomycin D treatment¹⁴⁾, indicating that this increase of VASH1 mRNA is mediated by the transcriptional activation.

Post-transcriptional regulation controls the fate of mRNAs at the steps of splicing, export, stabilization, and translation; and these processes are regulated by the interaction of cis-regulatory elements on mRNA and trans-acting factors such as RNA binding proteins (RBPs) and microRNAs that bind to these elements¹⁵⁾. When HUVECs are exposed to cellular stresses such as H₂O₂ treatment or serum starvation, HUVECs promptly increase the VASH1 protein level, and this is not accompanied by the increase of VASH1 mRNA. This prompt increase in VASH1 protein synthesis without transcription is mediated by an RBP, namely, HuR⁵⁾. HuR belongs to the embryonic lethal abnormal vision (ELAV) family of RBPs that bind to U-rich and/or AU-rich elements in the 3′UTRs of their target mRNAs, and it prevents their degradation and enhances translation¹⁵⁾. There are four members of the ELAV protein family, i.e., HuB, HuC, HuD, and HuR. HuB, HuC, and HuD are selectively expressed in the nervous system and play roles in neuronal differentiation and plasticity, whereas HuR is ubiquitously expressed and involved in numerous cellular responses¹⁵⁾. HuR is present predominantly in the nucleus under normal conditions. When cells are exposed to cellular stresses, HuR is translocated to the cytoplasm, where it interacts with mRNAs and elicits cellular stress responses¹⁶⁾.

Thus, VASH1 protein synthesis is induced transcriptionally for angiogenesis inhibition or post-transcriptionally for stress tolerance of ECs, and these functions are critical for the maintenance of vascular homeostasis (Fig. 1).

Fig. 1.

Regulation of VASH1 expression in EC

VASH1 is induced transcriptionally by angiogenic stimuli for angiogenesis inhibition or post-transcriptionally by RNA binding protein HuR for stress tolerance of ECs, and these functions are critical for the maintenance of vascular homeostasis. VASH1 is downregulated with replicative senescence of ECs by the increase of miR-22, and this age-associated downregulation of VASH1 might be a risk of deterioration of vascular homeostasis and age-related vascular diseases.

Age-Associated Downregulation of VASH1

It is well documented that a variety of functional classes of genes are downregulated with age, often in a tissue specific manner¹⁷⁾. We found that the expression of VASH1 was decreased with replicative senescence of ECs¹⁰⁾. To explore the mechanism of this downregulation of VASH1, the expressions of micro-RNAs in old HUVECs (49 population doublings) and young HUVECs (5 population doublings) were compared by performing microarray analysis¹⁰⁾.

MicroRNAs are small non-coding RNAs, which regulate gene expression at the post-transcriptional level via target mRNA degradation and/or translational suppression via binding to the 3′-untranslated region (3′-UTR)¹⁸⁾. It is estimated that approximately one-third of all animal genes are controlled by microRNAs, and thus microRNAs exhibit a wide range of biological processes including aging¹⁹⁾. Indeed, increasing evidences suggest that changes in the expression profile of microRNAs contribute to cellular senescence, aging and aging-related diseases, and several studies were directed to examine the relationship between certain microRNAs and cardiovascular aging^{20, 21)}. Our analysis revealed that among the top 20 microRNAs that were expressed at a higher level in old HUVECs, the third highest microRNA, namely, miR-22-3p, had its binding site on the 3′-UTR of VASH1 mRNA. Experiments with microRNA mimic and anti-miR revealed that miR-22-3p was involved in the downregulation of VASH1 in ECs during replicative senescence¹⁰⁾. MiR-22 is previously reported to promote senescence of endothelial progenitor cells by targeting AKT3²²⁾. We added VASH1 to the list of miR-22 targets that were downregulated with replicative senescence of ECs, and that might be responsive to aging-associated vascular diseases (Fig. 1).

The Decrease of Vash1 Gene for Healthy Longevity

Since VASH1 has a characteristic of promoting stress tolerance in ECs, we speculated that the lack of the Vash1 gene should result in a short lifespan. However, to our surprise, Vash1 KO mice lived significantly longer with a milder senescence phenotype than wild-type mice²³⁾.

Numerous studies have being focusing on the mechanism of aging, and so far they have revealed certain genes and signaling pathways that contribute to the regulation of the lifespan²⁴⁾. Among them, insulin signaling plays a crucial and evolutionally conserved role in longevity²⁵⁾. Decreased insulin signaling for longevity was first demonstrated in Caenorhabditis elegans, next Drosophila, and further confirmed in mammals^26–28). However, as insulin signaling is essential for glucose homeostasis, this decreased insulin signaling should cause diabetes mellitus. So-called insulin paradox has raised a challenging question as to how the preserved insulin signaling for glucose homeostasis and the reduced insulin signaling for longevity can be balanced²⁹⁾.

Skeletal muscle, liver, and adipose tissue are three major target organs of insulin for glucose homeostasis. We sought the cause of healthy longevity of Vash1 KO mice and found that Vash1 KO mice exhibited mild insulin resistance along with reduced expression of the insulin receptor (Insr), insulin receptor substrate 1 (Irs1), and insulin receptor substrate 2 (Irs2) in their white adipose tissue (WAT) but not in their liver or skeletal muscle. The expression of Vash1 dominated in the WAT among those three organs of WT mice, and its expression was selective in ECs. Importantly, Vash1 KO mice never developed diabetes when fed a high-fat diet²³⁾. This is the first demonstration that endothelial cell-expression of Vash1 in adipose tissue is required for the normal expression of Insr, Irs1, and Irs2, and insulin sensitivity of the WAT (Fig. 2). Although the decrease of Vash1 may be responsive to aging-associated vascular diseases, which also causes mild insulin resistance without the outbreak of overt diabetes and that may contribute to healthy longevity.

Fig. 2.

The decrease of Vash1 gene for healthy longevity

A: Reduced insulin signaling can increase longevity, but its profound reduction can result in diabetes and short life. The lack of Vash1 gene caused mild insulin resistance without the outbreak of overt diabetes and that might contribute to healthy longevity (asterisks). B: Skeletal muscle, liver, and adipose tissue are three major target organs of insulin for glucose homeostasis. The expression of Vash1 dominated in the vasculature distributed to WAT among three major target organs of insulin, and the expression of Vash1 is involved in the regulation of the expression of Insr, IRS1, and IRS2.

Concluding Remarks

This mini review summarized a molecular basis for vascular homeostasis and healthy longevity regulated by VASH1. VASH1 is a negative-feedback regulator of angiogenesis synthesized by ECs, which also increases stress tolerance of ECs. Besides its transcriptional induction by angiogenic stimuli, the synthesis of VASH1 receives post-transcriptional regulation. Cellular stresses increase the synthesis of VASH1 via HuR, whereas replicative senescence of ECs decreases the synthesis of VASH1 via an increase of senescence-associated microRNA, namely miR-22. As a decrease in VASH1 expression makes the vasculature vulnerable to cellular stresses, we were puzzled and wondered why nature would allow a decrease in the expression of such a valuable protein with aging. We then found that the lack of Vash1 gene resulted in health longevity in mice. In accordance with this observation, we now propose the novel role of VASH1 that influences the insulin signaling, and that one purpose of age-associated downregulation of VASH1 might be to afford healthy longevity (Fig. 3).

Fig. 3.

Double-face of VASH1 for the maintenance of vascular homeostasis and healthy longevity

The expression of VASH1 is maintained in reproductive period but downregulated during aging, and this age-associated downregulation of VASH1 might be a risk of deterioration of vascular homeostasis and age-related vascular diseases. VASH1 has an additional role that influences the insulin signaling, and another face of age-associated downregulation of VASH1 might be for healthy longevity.

COI

Nothing to declare.