Atherosclerosis is an underlying pathophysiologic disease of the arterial wall and a leading cause of death worldwide [1, 2]. Atherosclerosis is associated with chronic inflammation due to endothelial damage that contributes to the formation of atheromatous plaques in the arterial tunica intima. The developmental process of atheromatous plaques is slow and developed over several years through a complex series of cellular events occurring within the arterial wall and in response to various local vascular circulating factors. Atherosclerosis is the main cause of coronary artery disease (CAD) and stroke, which can happen when an atheromatic plaque ruptures and an atheroma is detached from it and blocks a blood vessel in the heart or the brain, respectively [1]. Moreover, atherosclerosis can lead to peripheral vascular disease [3] when the atheromatic plaque completely blocks the artery.

The relationship between hypoxia and endothelial dysfunction in atherosclerosis is complex and unclear [4]. Since many factors are involved in the signaling and action of hypoxia-inducible factor 1 alpha (HIF-1$\alpha$) and vascular endothelial growth factor (VEGF), they could be a target for atherosclerosis therapy. This systematic review article outlines the current evidence on the role of HIF-1$\alpha$ and VEGF immunophenotypes with other prognostic markers as potential biomarkers of atherosclerosis prognosis and treatment efficacy. Evidence of HIF-1$\alpha$ and VEGF protein expression using immunoassays in coronary artery cells, macrophages, or serum is presented, along with their potential involvement in atherosclerosis progression and atheromatic plaque vulnerability. Insights into the role of HIF-1$\alpha$ and VEGF immunophenotypes reflecting targeted therapy of atherosclerosis could suggest their use as potential biomarkers in the efficacy of atherosclerosis therapy.

Hypoxia, a condition in which the body’s tissues have insufficient oxygen supply, can result from several factors, including respiratory issues, cardiovascular problems, and cancer [5, 6], as discussed recently by our team [6]. Hypoxia triggers molecular mechanisms, such as stabilizing HIF-1$\alpha$, a protein subunit of the HIF-1 transcription factor, which plays a key role in the cellular response to low oxygen levels. When oxygen levels drop, HIF-1$\alpha$ is stabilized and translocated to the cell nucleus, where it forms a complex with another subunit, HIF-1$\beta$. This complex activates the transcription of genes involved in variable processes, such as angiogenesis, erythropoiesis, and glycolysis [7]. The impact of hypoxia, particularly HIF-1$\alpha$, is strongly associated with the pathogenesis of atherosclerosis [4]. Specifically, HIF-1$\alpha$ overexpression is associated with the formation, progression, and vulnerability of atherosclerotic plaques and inflammatory processes in atherosclerosis [4]. Therefore, HIF-1$\alpha$ is evolving as an attractive therapeutic target of atherosclerosis, with several promising research studies based on it in recent years.

The VEGFs represent a family of heparin-binding proteins that play a key role in multiple processes, including the pathways of angiogenesis, lymphopoiesis, and lymphangiogenesis, as well as in cells’ responses to oxidative stress and inflammation and the management of lipid metabolism [8, 9]. Its primary role is to stimulate the proliferation and growth of endothelial cells in all kinds of vessels. Moreover, VEGF can prevent the apoptosis of ischemic endothelial cells through the expression of various anti-apoptotic proteins. Hence, VEGF influences erythropoiesis, increases vascular permeability, and mediates inflammation with plural hemodynamic effects. Although VEGF has a key role in various physiological processes, the same properties make it play a role in the origin and maintenance of various pathological processes, including atherosclerosis [8].

There are five members of the VEGF family, including VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF) [9], involved in the regulation and differentiation of the vascular system, particularly in the blood and lymph vessels, with the most significant VEGF-A for its mediating angiogenic effects [8]. There are three receptors targeted by VEGF, such as VEGF receptor 1 (also VEGFR-1 or Flt-1) and VEGF receptor 2 (also VEGFR-2, Flk-1, or KDR), mainly expressed in vascular endothelial cells (VECs) and VEGFR3, found in lymphatic endothelial cells (LECs) [9]. VEGF-A can be expressed in the cardiovascular system and other tissues under conditions of inflammation and hypoxia. Its secretion is upregulated by hypoxia-inducible factors (HIF), specifically by HIF-1$\alpha$, and downregulated by low-density lipoprotein (LDL) and lipid-lowering drugs (rosuvastatin). It has been previously discussed that VEGF-A prevents the repair of an endothelial lesion and induces the expression of adhesion molecules in endothelial cells, stimulating the secretion of chemokines, which recruit monocytes to migrate into the blood vessel wall and promote endothelial permeability [9]. These monocytes accumulate oxidized LDL and transform into foam cells. VEGF-A additionally promotes the migration of vascular smooth muscle cells (VMSCs) into the plaque, which could lead to platelet activation, aggregation, and thrombus formation [9]. VEGF-A binds to and activates VEGFR-1 and VEGFR-2; the latter is mainly associated with pathological angiogenesis, including vessel formation in tumors [8]. VEGF-B can be found in many tissues, primarily the cardiovascular system, kidney, fat, lung, pancreas, and gallbladder. VEGF-B can only bind to VEGFR-1 and promote angiogenesis, with a substantial impact on neovascularization in the myocardium after a myocardial infarction (MI), and reduces oxidative stress to VECs. Additionally, it has a strong anti-apoptotic effect on myocardial cells after MI, inducing the uptake of fatty acids by endothelial cells, thus lowering the lipid levels in blood serum [10]. VEGF-C and VEGF-D share structural and functional homology to a greater degree than with the other VEGFs. Specifically, VEGF-C is highly expressed in embryonic tissues but also expressed in an adult’s organs. VEGF-C binds to VEGFR2 and VEGFR3, and its main function is regulating and shaping the lymphatic network. Moreover, VEGF-C has an angiogenetic, fibrogenetic, anti-apoptotic, and lipid-increasing effect [10]. VEGF-D is found mainly in the lung (embryonic and adult stages), heart, and intestine. VEGF-D also binds to VEGFR-2 and VEGFR-3 and induces lymphangiogenesis, angiogenesis, fibrogenesis, and a lipid-lowering effect [9, 10]. In addition, the placental growth factor (PlGF) is another member of the VEGF family, which binds to VEGFR-1 [8].

HIF-1$\alpha$ and VEGF are strongly correlated through the same metabolic path, affecting angiogenesis and atherosclerosis [6, 10]. Under hypoxia, HIF-1$\alpha$ activates the secretion of VEGF to promote angiogenesis, a higher blood and O₂ supply, and cell adaptation to hypoxia. HIF-1$\alpha$-induced VEGF contributes to vascular remodeling, which can be classified as positive or negative. HIF, VEGF, and cytokines are primarily found in positively remodeled vascular plaques, which contain more macrophages, larger necrotic cores, and thin fibrous caps and, thus, are more prone to breakage [11]. On the other hand, negatively remodeled plaques are more stable [11].

Atherosclerosis results from various interactions between malfunctioning endothelial cells and smooth muscle cells, lipid aggregation, inflammation, calcification of the atheromatic plaques, degradation of the extracellular matrix, altered hemopoiesis, and genetic predispositions [12, 13, 14]. Specifically, the dysfunctional endothelium combined with the accumulation of lipids within the arteries and their effect on the tunica intima are the main initiators of atherosclerosis [12]. Various inflammatory cytokines are released, leading to the activation of inflammatory pathways responsible for the development of the fatty streak, the first evidence of atherosclerosis [12]. As more lipids are deposited in the plaque, more inflammatory cytokines are released, more monocytes are recruited, and the inflammation becomes chronic.

Several risk factors have been implicated in atherosclerosis (Table 1, Ref. [1, 2, 3, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21]). In general, atherosclerosis is described as a preventable lifestyle disease. Hypercholesterolemia is the primary cause of atherosclerosis [14]. Mutations in the low-density lipoprotein receptor (LDLR) gene, as well as mutations in other genes, including the Apolipoprotein B (APOB) gene, proprotein convertase subtilisin/kexin type 9 (PCSK9) gene, and low-density lipoprotein receptor adaptor protein 1 (LDLRAP1) gene have been found in individuals with familial hypercholesterolemia, a hereditary form of hypercholesterolemia [13, 15, 16]. Nutrition and obesity have been linked to the development of atherosclerosis [1, 14]. Smoking, recurrent exposure to environmental and psychological stressors, as well as inadequate or poor-quality sleep, are also risk factors for the development of atherosclerosis [12, 14]. The mechanism underlying the aforementioned factors has been linked to the activation of the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis. This activation leads to low-grade inflammation, which in turn affects the functioning of the endocrine and vascular systems [14].

Atherosclerosis is a chronic inflammatory condition defined by the buildup of lipids, fibrous tissue, and calcification inside large and medium-sized arteries [17] (Table 1). Accumulating evidence suggests that the inflammation surrounding the atheromatic plaque links the various risk factors and the development of atherosclerosis [1, 3, 12, 18, 19]. A growing body of research has proposed that pro-inflammatory substances, such as cytokines, are secreted and accumulated within the arteries due to the oxidation of the lipoproteins that are present in the blood, and this, in turn, leads to the recruitment of white blood cells (mainly monocytes and macrophages, but also T-cells and B-cells in the area) and consequently inflammation [1, 2, 12, 14]. Other researchers do not embrace this hypothesis because using antioxidant medicines has not managed to decrease atherosclerosis. Therefore, they have proposed different mechanisms regarding the presence of inflammation, including accumulated LDL [1], while various experimental studies have provided evidence in favor of the lipid oxidation hypothesis and have managed to do this by manipulating different variables, including transgenic expression of antibodies in animals or by targeting specific receptors implicated in the processing of lipids [1]. In addition, several parameters have been parallelized to plaque pathogenesis, such as LDL/(high-density lipoproteins) HDL levels, (apolipoprotein-AI) Apo-AI levels, triglycerides, oxidized low-density lipoprotein (ox-LDL), and inflammation mediators, which are also used as biomarkers, such as C-reactive protein (CRP) [20].

Regarding the histopathological findings of atherosclerosis, it has been reported that coronary arteries show signs of calcification in the tunica media, a “heart” full of lipids and surrounded by a fibrous layer and numerous foam cells [12, 21]. Features of ulceration and thrombosis can be found, as well as intimal thickening, narrowing of the lumen, and stenosis of various degrees (Table 1). Severe stenosis in large arteries can be identified macroscopically, but a smaller degree requires a light microscope.

Atheromatic lesions are either fibrotic or abundant in foam cells by using hematoxylin-eosin staining or monoclonal antibodies against various antigens (immunohistochemical procedures) [21] (Table 1). Specifically, histologically, the atheromatous plaque is characterized by the accumulation of monocytes, macrophages/large foam cells with high lipid content, and smooth muscle proliferation in response to cytokines secreted by damaged endothelial cells (Table 1).

We used PRISMA to report our systematic review and ROBIS (Risk of Bias in Systematic) as a quality assessment tool for the risk of bias, as follows:

2.4 Synthesis and Findings

Our search revealed 34 full-text articles (Fig. 1) by PRISMA 2020 [22], including 650 human specimens, 21 different cell lines, and 9 different animal models. Most of these articles (53%), particularly those that discussed HIF-1$\alpha$ and VEGF-related immunophenotypes in atherosclerosis-targeted therapy (76%), were published between 2019 and 2024. The main details of the studies about the HIF-1$\alpha$/VEGF immunophenotypes in atherosclerosis are shown in Tables 2,3,4 and summarized in Figs. 2,3,4,5.

Fig. 1.

The diagram shows the articles included in this study (by PRISMA 2020).

It is worth mentioning that based on our search, a great volume of atherosclerosis-related articles was published from 2009 to May 2024 (PubMed: 112,414; Embase: 99,856; Medline: 178,200), showing a health emergency due to the size of the disease.

Atherosclerosis is associated with chronic inflammation due to endothelial damage leading to the formation of atheromatous plaques. HIF-1$\alpha$ regulates inflammation, angiogenesis, response to oxidative stress, and development of foam cells [60]. Also, HIF-1$\alpha$ adjusts the pathophysiology of macrophage foam cells [61, 62]. Here, we performed a systematic review to show the association of HIF-1$\alpha$ and VEGF immunophenotypes in atherosclerosis prognosis and targeted therapy. Despite the increasing interest in hypoxia and atherosclerosis, especially because of its therapeutic perspective, the relevant literature was not extensive. We found 34 preclinical and clinical studies proving the association of HIF-1$\alpha$ and VEGF immunophenotypes with atherosclerosis prognosis or the effectiveness of therapy. Data limitations of this study may include the use of different survival methods. Our data are summarized in Fig. 6. In general, elevated HIF-1$\alpha$ and VEGF levels in coronary artery cells and macrophages are strongly associated with poor prognosis of atherosclerosis. Since data of elevated HIF-1$\alpha$ and VEGF have also been reported to correlate with a favorable prognosis, therapeutic approaches that either reduce or stabilize HIF-1$\alpha$ and VEGF immunophenotypes are applied for the attenuation of atherosclerosis.

Fig. 6.

The Role of HIF-1$\alpha$ and VEGF as Biomarkers in the Prognosis and Evaluation of Treatment Efficacy of Atherosclerosis. Prognosis: Hypoxic-induced increased HIF-1$\alpha$ and VEGF immunophenotypes in vascular smooth muscle cells (VSMCs), macrophages, or endothelial cells are associated with glycolytic flux, lipid accumulation, vessel narrowing, and necroptosis, inducing increased inflammation and angiogenesis, leading to atherosclerosis progression. However, increased levels of HIF-1$\alpha$ and VEGF found in lymphocytes (CD4+ spleen-derived T-cells) are associated with attenuated atherosclerotic lesions. Similarly, increased levels of HIF-1$\alpha$ with stable VEGF overexpression in macrophages or plasma contribute to the attenuation of intimal hyperplasia after arterial injury and a favorable prognosis for atherosclerosis. Targeted therapy: Targeting HIF-1$\alpha$ using PX-478, fluvastatin, a prolyl hydroxylase domain-containing protein 2 (PHD2), Buyang Huanwu decoction (BHD), or Crocin can suppress hypoxia-induced HIF-1$\alpha$ immunophenotype in coronary artery cells, endothelial cells, or macrophages or reduce cholesterol levels in serum, preventing atherosclerosis. Similarly, the application of HIF-prolyl 4-hydroxylases (HIF-P4Hs) inhibitor, FG-4497, can stabilize HIF-1$\alpha$ expression and reduce cholesterol in serum, protecting against the development of atherosclerosis. In parallel, targeting VEGF using adenosine receptors (AR), polyphenols (anthocyanins), notoginsenoside R1 (NR1), 7-difluoromethoxy-5,4^′-dimethoxygenistein (DFMG), glycosides, tetramethylpyrazine (TMP), and paeoniflorin (PF) or methylated regulation (METTL3) in endothelial cells can regulate hypoxic-related atherosclerotic progression by decreasing the levels of inflammation, reducing atheromatique plaque pathology, or alleviating vascular endothelial cell injury. However, the inhibition of lncRNA NORAD can increase VEGF immunophenotypes in endothelial cells, alleviating ox-LDL-induced vascular endothelial cell injury and atherosclerosis development. Similarly, inhibition of miR-497-5p can alleviate ox-LDL-induced downregulation of VEGFA and dysfunction of endothelial cells, contributing to atherosclerosis prevention.

We present that hypoxia-induced elevated HIF-1$\alpha$ and VEGF expression in smooth muscle cells can be found in early-stage atherosclerosis development [24], accompanied by increased levels of a proinflammatory pathway of NF-$\kappa$B, CRP, and IL-6 [26]. Kimura et al. [63] previously discussed that serum VEGF may be an arteriosclerosis-promoting cytokine in humans and proven that elevated CRP and VEGF can be detected earlier than the development of arteriosclerosis in smokers.

We also show that HIF-1$\alpha$ overexpression induces increased MIF expression, which mediates smooth muscle cell migration and proliferation and, thus, is linked to vessel narrowing and atherosclerosis progression [29]. Similarly, Kastora et al. [64], in a review article, discussed data from animal studies, presenting that upregulation of VEGF decreases lumen stenosis and neointimal hyperplasia, both protective factors against atherosclerosis.

Moreover, we present that elevated HIF-1$\alpha$ expression induces transcriptional activation of HILPDA in macrophages, which mediates lipid accumulation and plaque formation [25]. In addition, we show that elevated HIF-1$\alpha$ expression in human endothelial cells due to its accumulation resulting from chronic loss of NO can lead to modifications to endothelial cell physiology and dysfunction and the progression of atherosclerosis [30]. Under hypoxic conditions, elevated levels of HIF-1$\alpha$, hexokinase II, and PFKFB3 are also associated with enhanced proinflammatory activity and macrophage glycolytic flux, which affect macrophage viability [28]. However, increased HIF-1$\alpha$ levels in mouse lymphocytes have also been associated with a favorable prognosis due to the direct effect of HIF-1$\alpha$ on the lymphocytic cytokine profile, causing a significant reduction in IFN-$\gamma$, the transcription of, and attenuation of atherosclerosis [27].

Regarding genetics, we show that increased levels and activity of HIF-1$\alpha$ in hypoxic-induced macrophages can be negatively regulated by miR-17 and miR-20a. This can influence the differentiation from monocytes to macrophages in the progression of atherosclerosis [31]. Furthermore, we show that elevated levels of HIF-1$\alpha$ in inflammatory macrophages can increase necroptosis via microRNA-mediated ATP depletion, leading to atherosclerosis and plaque vulnerability [32].

As mentioned above, HIF-1$\alpha$ and VEGF, primarily induced by HIF-1$\alpha$, strongly correlate through the same metabolic path, affecting angiogenesis and atherosclerosis [65]. Induced by HIF-1$\alpha$, VEGF-A primarily has either harmful or beneficial effects on atherosclerosis. Earlier studies have shown that hypoxia and inflammation trigger VEGFA’s secretion in VMSCs and macrophages [9, 65, 66, 67]. In addition, it has been previously shown that VEGF contributes to vascular remodeling [11]. HIF, VEGF, and cytokines are primarily found in positively remodeled vascular plaques, which contain more macrophages, larger necrotic cores, and thin fibrous caps and, thus, are more prone to breakage, while negatively remodeled plaques are more stable [11].

Based on our data, HIF-1$\alpha$-induced VEGF correlates with a higher CAD severity by mediating the growth and breaking of plaques, the latter due to the activation of the influx of inflammatory cells and erythrocytes [33]. Specifically, we present that elevated VEGF levels in coronary artery cells are associated with serum IL-18 levels and a higher CAD severity, and thus, VEGF may be involved in CAD pathophysiology [33]. We show that chronic exposure to METH can induce elevated levels of VEGF in the coronary artery that mediate angiogenesis and vessel rupture in atherosclerotic plaque [34]. Moreover, we show that elevated VEGF in macrophages or endothelial cells can activate, via p38$\alpha$ phosphorylation, both LITAF and STAT6B to further upregulate VEGF, angiogenesis, and atherosclerosis [35]. Hypoxia and inflammation trigger VEGFA’s secretion in VMSCs and macrophages [9]. Here, we present that increased HIF1$\alpha$ and VEGF-A expression in CD163+ macrophages is associated with plaque progression [36], highlighting a nonlipid-driven mechanism of angiogenesis via the CD163/HIF1$\alpha$/VEGF-A pathway.

On the contrary, we present preclinical and clinical data that support a correlation of reduced risk of atherosclerosis when VEGF levels are stable. Specifically, we present clinical data [37, 38] showing that circulating VEGF (VEGF-A) levels are only marginally associated with a considerable risk of atherosclerosis. In addition, we present in vitro data showing that stable overexpression of VEGF correlates with reduced macrophage foam cell formation and down-regulation of CD36 expression in macrophages, attenuating the progression of atherosclerosis [39]. Similarly, we present in vitro and in vivo data that stable overexpression of VEGF can promote the expression of anti-apoptotic proteins and produce high NO levels, contributing to the attenuation of intimal hyperplasia after arterial injury [40]. However, the association of stable levels of VEGF in blood with a higher or lower risk of atherosclerosis needs further clarification. Moreover, based on data from animal models and humans treated for cancer, anti-angiogenic factors, including blocking VEGF-A and the signaling downstream from their receptors, may reduce the formation of atheromatic plaques [68, 69].

A mechanism by which HIF-1$\alpha$ and VEGF interact under hypoxia and their association with angiogenesis have been previously proposed [64]. This mechanism may include oxidative stress through heat shock protein 27 (HSP27), tissue trauma, and inflammation through activator protein 1 (AP-1), caveolin-1 (Cav-1), or nitric oxide synthase (NOS). VEGF-A bindings to VEGFR2 may lead to downstream proangiogenic cascades [52]. In addition, cross-activation of the VEGF receptors by multiple VEGF family members may lead to variable effects [70]. Another mechanism is the promotion of the production of VEGF by HIF-1 connection to the HRE in the VEGF promoter region. VEGFR-1 and VEGFR-2 are the two main receptors expressed on endothelial cells, mediated by HIF-1. Under hypoxia, HIF upregulates VEGFR-1 directly by binding to the enhancer element in the VEGFR-1 promoter, while VEGFR-2 is post-transcriptional upregulated [41, 71]. Based on a molecular mechanism proposed by Chen et al. [72], HIF-1$\alpha$ and VEGF interaction may lead to negative regulation of atherosclerosis. This mechanism shows silent information regulator 1 (SIRT1) to negatively regulate atherosclerotic angiogenesis via mammalian target of rapamycin 1 (mTORC1) and HIF-1$\alpha$ signaling that may result in a decreasing VEGF secretion in atherosclerotic plaques [72]. Also, a computational mechanistic model created by Zhao & Popel [73] may shed some light on the molecular control of the HIF-1/VEGF pathway. Based on this model, under hypoxia, members of the let-7 miR family are identified as hypoxia-responsive miRNAs (HRMs) whose levels are robustly upregulated by HIF-1. Mature miR-7 targets argonaute family 1 (AGO1) mRNA, essential for miRNA function. This leads to the downregulation of AGO1 and other miRNAs that target VEGF, thus freeing VEGF from translational repression to promote angiogenesis [73].

The above observations may explain our data summarized in Fig. 6, showing that increased HIF-1$\alpha$ and VEGF immunophenotypes may have a different prognosis in atherosclerosis depending on the cellular environment and conditions, as well as on the VEGF subtype analyzed due to complex interactions between VEGFs and VEGFRs. Based on our data, VEGF inhibition can prevent hypoxia-associated atherosclerosis through the negative regulation of the Ang1-Tie2/PI3K-AKT or TLR4/NF-$\kappa$B/VEGF signaling pathway, the latter using small regulatory molecules such as miR-140 (Fig. 5).

How HIF-1$\alpha$ and VEGF are correlated under different cellular environments and pathological conditions, such as inflammation and tumor angiogenesis, and how they correlate with factors that may influence their different pathological effects on atherosclerosis have been previously discussed [9, 64, 68, 74]. Under hypoxic conditions, HIF-1$\alpha$ activates the secretion of VEGF and other transcriptional factors [66], which promotes angiogenesis, a higher blood and O₂ supply, and cell adaptation to hypoxia [9, 11]. HIF-1$\alpha$ and VEGF-A are both increased in the early phase of ischemia or infarction; however, persistent oxygen deficiency may hinder the production of angiogenic factors due to widespread cell deaths [75]. Moreover, dysregulation of vascular homeostasis in atherogenesis during aging may include VEGF downregulation, which is strongly correlated with the decreased levels of P-STAT3 (phospho-STAT3) and P-CREB (phospho-cAMP response element-binding protein) in association with HIF-1$\alpha$ [65, 66]. In addition, the upregulation of HIF-1$\alpha$ and VEGF has been associated with cytokine networks and apoptosis [76]. Based on a proposed mechanism, caspase 1/interleukin 1-$\beta$ may interact with HIF-1$\alpha$ and VEGF during retinopathy damage to the blood vessels [76]. Specifically, hypoxia-induced activation of HIF-1$\alpha$ stimulates VEGF production, which leads to angiogenesis, and vasculogenesis may, in parallel, lead to caspase-1 and an acute inflammatory response. Caspase-1 is a cysteine protease that responds to apoptotic stimuli and can cleave precursors to inflammatory cytokines into active forms. This results in activating and releasing pro-inflammatory molecules, such as IL-1$\beta$, and activation of programmed cell death. Blocking the caspase-1/IL-1$\beta$ signaling cascade can inverse this process. Although it is not clear yet how the presence of VEGF-A may affect caspase 1, IL-1$\beta$, and HIF-1$\alpha$ signaling within retinal capillaries, it is suggested that HIF-1$\alpha$ and VEGF may affect retinal capillaries.

In addition, HIF-1$\alpha$ and VEGF have also been reported to play a significant role in tumor angiogenesis [77]. More specifically, when cancer cells proliferate, forming a solid tumor, there is an imbalance between oxygen supply and oxygen demand. Therefore, newly activated HIF-1$\alpha$ is not ubiquitinated and is targeted to proteasomal degradation. Activated HIF-1$\alpha$ upregulates the expression of various proangiogenic genes, such as VEGF, and their receptors, such as VEGFR1, also known as Flt-1 or Flk-1, as well as Ang1, Ang2, and Tie2. VEGF is considered a primary effector of tumor angiogenesis via “angiogenic switching, in which tumor cells stimulate tumor progression through angiogenesis induction by supplying oxygen and nutrients through the newly created capillaries. A hypoxia-independent stimulation of HIF-1$\alpha$ in solid tumors through antioncogenes’ alterations has also been described [77]. Similar findings are also seen in studies testing anti-HIF agents in anti-angiogenesis therapy in carcinoma [78, 79, 80].

The potential therapeutic implications of targeting HIF-1$\alpha$ for atherosclerosis have been previously discussed [41]. Here, we show that HIF-1$\alpha$ immunophenotypes are reduced after PX-478 application, which is an inhibitor of HIF-1$\alpha$, resulting in reduced cholesterol levels in plasma and reduced atheroma, directly affecting the development of atherosclerosis [43]. Furthermore, we present that CNP endogenous peptide promotes HIF-1$\alpha$ degradation, enhancing plaque stability, while LCZ696, a drug, can amplify the bioactivity of CNP and ameliorate atherosclerotic plaque formation [44]. In addition, we show that HIF-1$\alpha$ expression is reduced in the coronary artery wall by statins [45]. Specifically, we found that HIF-1$\alpha$ expression and its dependent ET-1 expression are reduced in VSMCs by fluvastatin application, resulting in the attenuation of atherosclerosis [45]. On the other hand, we present that HIF-P4As inhibition can stabilize HIF-1$\alpha$ expression, reducing serum cholesterol and inflammation and protecting against the development of atherosclerosis [46]. Finally, we show that natural products, such as BHD, a classic herbal formula of traditional Chinese medicine, or crocin, can inhibit HIF-1$\alpha$ expression in aortic tissue to prevent atherosclerosis [47, 48].

Due to the direct relation of HIF-1$\alpha$ with VEGF, it has been previously discussed that the application of angiogenesis inhibitors may improve plaque stability and reduce plaque progression. Thus, several drugs targeting VEGF are being processed in the preclinical and clinical stages; however, anti-angiogenic therapy is still debatable because of the side effects on vasculature [8, 68]. About VEGF immunophenotypes, we show that VEGF is reduced in endothelial cells through the application of polyphenols, methylation modification, antagonists of adenosine receptors, or natural products [49, 50, 51, 52, 53, 54, 55]. Specifically, we show that VEGF and IL-8 expression is reduced by adenosine receptor antagonists blocking important steps in adenosine-mediated atherosclerotic plaque development [49]. In addition, we show that reduced VEGF levels are induced by polyphenols, such as anthocyanins, resulting in restriction of angiogenesis, glycosides, and atherosclerotic progression [50]. Similarly, we present that VEGF is reduced in endothelial cells after METTL3 knockdown, preventing atherosclerosis [51]. Moreover, we present natural products with anti-atherosclerotic effects, such as the combination of TMP and PF, and NR1, an herbal monomer isolated from Panax notoginseng (Burk.) F. H. Chen, can reduce the expression of VEGF levels in arterial endothelium [52, 53, 54]. Specifically, we show that the downregulation of protein expression of VEGF and angiogenesis-related factors, including Notch1, in TMP and PF-treated cells contributes to the increase of plaque stability [52]. Triptolide (TPL) has also previously been discussed by others as another natural compound that mediates the downregulation of eNOS, VEGF, and VEGFR2 and could limit atherosclerotic plaque enlargement [9].

Here, we present that NR1 can induce a reduced VEGF immunophenotype, decrease the levels of inflammatory factors [53], and induce a noticeable reduction in plaque pathology by suppressing the Ang1-Tie2/PI3K-AKT paracrine signaling pathway [54]. We also show that DFMG, a new chemical entity synthesized with genistein, a natural phytoestrogen, can inhibit VEGF protein levels, mediated by miR-140 negative regulation of the TLR4/NF-$\kappa$B signaling pathway, showing anti-atherosclerotic effects [56]. Furthermore, we present that the expression of HIF-1$\alpha$ and VEGF can be abolished in cells treated with a combination of glycosides through STAT3 inhibition, preventing atherosclerosis [55]. On the other hand, we show that the inhibition of lncRNA NORAD can reverse VEGF-reduced immunophenotype, alleviating vascular endothelial cell injury and atherosclerosis development [57]. Finally, we show that miRNAs may play a role in therapeutic approaches for atherosclerosis. Specifically, we present that inhibition of miR-497-5p can attenuate ox-LDL-induced downregulation of VEGFA and dysfunction of endothelial cells through the p38/MAPK pathway [58].

In addition to the data presented here, earlier studies also discussed the effect of VEGF on specific anti-VEGF drugs, such as monoclonal antibodies against the VEGF signaling pathway (VSP), VEGF-modified macrophages, or soluble forms of VEGF receptors, to treat atherosclerosis. VEGF-modified macrophages seem to decrease lipid accumulation, which reduces cell foam formation [9, 39]. Also, soluble forms of VEGF receptors have been previously discussed to improve the effects on atherosclerotic plaques by binding to VEGF and regulating its concentration in the plaque area [64]. Furthermore, VSP monoclonal antibodies can inhibit VEGF actions, influencing the survival and proliferation of endothelial cells and VSMCs [9]. However, they are also connected with cardiotoxic side effects [9]. Finally, melatonin has been previously discussed as a therapeutic target in atherosclerosis through VEGF [63]. Specifically, a recent study showed that melatonin can significantly and dose-dependently inhibit VEGF-induced angiogenesis in endothelial cells as well as attenuate smoking-induced atherosclerosis by activating the nuclear factor erythroid 2-related factor 2 (NRF2) pathway in endothelial cells [68].

In conclusion, hypoxia is involved in atherosclerosis, and the role of HIF-1$\alpha$ and its induced VEGF as potential biomarkers in the prognosis and evaluation of treatment efficacy of atherosclerosis has been thoroughly studied with experimental and clinical data. Increased levels of HIF-1$\alpha$ and VEGF in coronary artery cells or macrophages are associated with proinflammatory cell production, leading to atherosclerosis via increased glycolytic flux, lipid accumulation, vessel narrowing, necroptosis, and atheromatic plaque break. Chronic loss of NO or specific miRNAs can also regulate this process. However, overexpression of HIF-1$\alpha$ in lymphocytes or overexpression of VEGF in macrophages is associated with reduced foam formation and hyperplasia after arterial injury, attenuating atherosclerosis development or progression. In parallel, HIF-1$\alpha$ or VEGF immunophenotypes are altered in coronary artery endothelial cells or macrophages after the application of atherosclerosis-targeted therapy by using HIF-1$\alpha$ inhibitors, HIF-P4H inhibitors, statins, polyphenols, lnRNAs, methylation modification, adenosine receptor antagonists, natural products, or miRNA blockers, supporting the disruption of the HIF-1$\alpha$/VEGF pathway as an effective approach in atherosclerosis treatment.

Although the development of proper methods to accurately evaluate hypoxia in atherosclerotic lesions is still a challenge for the future, meta-analyses, including similar enough research studies, may reveal the clinical or methodological heterogeneity from various sources exploring the role of HIF-1$\alpha$/VEGF as a biomarker for the risk of atherosclerosis and treatment efficacy. However, a growing body of data supports the involvement of HIF-1$\alpha$ and its related VEGF in various atherosclerosis aspects. Further investigation in large clinicopathological experimental models and validation of the predictive value of HIF-1$\alpha$ expression in atheromatous plaque regulation is warranted, which may contribute to novel personalized treatment strategies in clinical practice. It is expected that such large-scale clinical studies, including anti-HIF or anti-VEGF therapeutic strategies and similar methodological approaches for the qualification and quantification of HIF-1$\alpha$ and VEGF immunophenotypes, will provide evidence of HIF-1$\alpha$ and VEGF profiles as a reliable and valuable clinical tool for detecting, prognosis, and monitoring patients with atherosclerosis.

HIF-1$\alpha$, Hypoxia-inducible factor 1 alpha; VEGF, vascular endothelial growth factor.

All data generated or analyzed during this study are included in this published article and are available from the corresponding author upon reasonable request.

Study conception and design: DPV, MI, KPM; literature search: DPV, PGD, AP, DG, MP, NL; data analysis: DPV; interpretation of data and results: DPV, PGD, AP, DG, MP, NL, KZ, SGD, KPM, MI; draft manuscript preparation: DPV, PGD, AP. All authors contributed to editorial changes in the manuscript. All authors reviewed the results and approved the final version of the manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Not applicable.

Not applicable.

This research received no external funding.

The authors declare no conflict of interest.

Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.31083/FBL27004.