Vasa vasorum in plaque angiogenesis, metabolic syndrome, type 2 diabetes mellitus, and atheroscleropathy: a malignant transformation

In 2003, Purushothaman KR et al. have presented data that neovascularization is the most powerful independent predictor for plaque rupture in vulnerable plaques (p= 0.0001) followed by disruption of the internal elastic lamina (p= 0.01), and fibrous cap thinness (p= 0.02) [21]. This exciting publication has also prompted this current review and has extended the important role of the adventitia and plaque angiogenesis in our understanding of remodeling of the arterial vessel wall in macrovascular disease and the development of the vulnerable plaque in MS, PD, and T2DM.

Just as atheroscleropathy has been used to describe the accelerated atherosclerotic process, the term intimopathy may be used to describe the accelerated positive outward remodeling and neovascularization of the intima and media in MS, PD, and T2DM. This neovascularization is derived from the adventitial vasa vasorum (Vv) and may also be termed intimal microangiopathy, which is similar to the retinopathy found in the retinal vascular bed.

The Vv is called into the intima and media by a process of angiogenesis, which is increased in MS, PD, and overt T2DM. As can be seen from table 1 this occurs at an accelerated rate in stages IV and V (table 2). There are at least three mechanisms driving the angiogenic response in atheroscleropathy and intimopathy – intimal microangiopathy associated with MS, PD, and T2DM:

1. The initial angiogenic response by the adventitial Vv (via angiogenesis) is stimulated by hypoxia and ischemia as the intima and media undergoes thickening (exceeding the ability of simple diffusion of nutrients from the lumen [usually greater than 0.35 millimeters]) and positive outward remodeling [13]. This hypoxia induces the adventitial Vv to undergo angiogenesis due to Hif-1 and VEGF stimulation [22].

2. In MS and T2DM there exists a microangiopathy within the adventitial derived Vv microvessels. There is increased vascular permeability (leaky micro-vessels) of red blood cells (RBCs) and plasma proteins (comparable to the proteinuria in diabetic nephropathy) into the interstitium of the ECM due to the microangiopathy, which instigates an inflammatory response of monocytes and macrophages [6, 7, 23, 24]. The inflammatory response is excessive in MS, PD, and T2DM and is further increased by the inflammatory signals induced by the increase of RBC plasma membrane extravasation. The inflammatory macrophages are a source of VEGF and other positive regulators of angiogenesis.

3. As the numbers of angiogenic vessels (Vv) increase, the probability of intraplaque hemorrhage increases with extravasated RBCs. The adventitial Vv microvessels within the atherosclerotic plaque are very fragile and prone to rupture as previously described. The inflammatory infiltrate runs concurrently with the development of the intimopathy (neovascularization) as oxidized LDL cholesterol, glycated LDL-cholesterol, and glycoxidated LDL-cholesterol (all of which are forms of modified LDL-cholesterol) also induce an antigenic stimulus and calls in the inflammatory macrophage, which is known to accumulate within the shoulder region of vulnerable atherosclerotic plaques.

Kolodgie FD et al. have just published an important article revealing the importance of the RBC plasma membranes and their deposition within the vulnerable atherosclerotic plaques [25]. The RBC plasma membrane deposition within the necrotic core, in addition to, free cholesterol, macrophage infiltration, and enlargement of the necrotic core is currently felt to be an additional atherogenic stimulus and concurrently increases the risk of plaque destabilization and rupture with ensuing ACS. This new information contributes to the importance of plaque angiogenesis, IPH, and aid in the understanding of the important role they play in the development of ACS.

The inflammatory infiltrates of the vulnerable shoulder region are responsible for:

1. Recruitment of more monocytes (fueling the inflammatory process).

2. Synthesis of tissue ACE and Tissue Factor (TF), which are known to be direct positive regulators of angiogenesis.

3. Promoting a thinning of the fibrous cap. (IFN gamma of the t-lymphocytes). [26]

4. Increasing the apoptosis of the smooth muscle cell (SMC). (IFN gamma of the t-lymphocyte). [26]

5. Promoting the increased modification of LDL-cholesterol.

6. Promoting the arrival of the t-lymphocyte to the shoulders of the plaque.

7. TNF alpha, IL-1, and IFN gamma by the t-lymphocyte.

8. An increased synthesis of VEGF and FGF.

Summary: 1. Tissue Factor. 2. Tissue ACE. 3. MMPs. 4. Cytokines and growth factors (figure 3) [6].

It is interesting to note that while there is synergism between the vasa vasorum and the inflammatory infiltrate there exists an opposing phenomenon between the SMC and the macrophage. The SMC is initially damaging (causing a progression of the atheroma and atherosclerotic plaque through fibroproliferation) and later in the atherosclerotic process becomes protective providing the strength of the fibrous cap [27]. The macrophage is initially protective (removing or modifying the injurious stimuli i.e. modified LDL-cholesterol to promote healing) and later becomes important in accelerating the disease process (the inflammatory process) by promoting instability and plaque rupture.

The vasa vasorum acts as a custom-delivery system for the following substrates:

1. Substrates of the Renin Angiotensin Aldosterone System.

2. Substrates of native LDL-cholesterol and modified LDL-cholesterol.

3. Substrates for angiogenesis. The endothelial progenitor cells, as well as, the endothelial cell itself via migration associated with the 10-point process of angiogenesis (table 2) (figure 5).

4. Supply the route for the second wave of inflammatory cells to the intima and vulnerable shoulder regions of the plaque. Fueling the inflammatory process. The first wave originating from the endothelial luminal surface.

5. Supply the direct route for the adventitial fibroblasts and TGF-beta allowing for intimal plaque expansion.

During the past few years there have been important articles published concerning the importance of angiogenesis within the unstable atherosclerotic plaque. Kwon reported (with micro computer tomography in the coronary porcine model) rapid, extensive development of vasa vasorum angiogenesis in coronary vessels. These were induced by a high fat diet in three months [28, 29]. Burke and Virmani revealed that plaque rupture in sudden death is related to vasa vasorum angiogenesis in addition to the chronic macrophage inflammatory response [30]. O'Brien was able to show that adhesion molecules (VCAM, ICAM and E-Selectin) expression was in the endothelial lining of the angiogenic vasa vasorum [31]. Thus, later in the atherosclerotic process the angiogenic vasa vasorum may be more responsible for the delivery of the previously discussed harmful molecules and substrates than diffusion through the traditional endothelial lumen surface early in the atherosclerotic process.

Recently de Boer and van der Wal [32] substantiated the above by staining for endothelial cells within the vulnerable plaque and concluded that angiogenesis and expression of adhesion molecules by the vasa vasorum may sustain the influx of inflammatory cells with subsequent plaque destabilization. They as O'Brien found that E-Selectin, ICAM and VCAM were up regulated. In addition they found the expression of CD40 to be increased. This custom delivery system of angiogenic vessels (Vv) acts to deliver the harmful substrates directly to the vulnerable plaque in addition to supplying a route for the chronic inflammation.

This is quite reminiscent of the angiogenic vessels noted in malignant tumors, which provides nourishment for the exponential growth and a vascular route for the metastasis of tumor cells to other organs.

In summary, atherosclerosis is malicious, resistant to treatment, occurring in severe form, frequently fatal, locally invasive, and destructive with growth, resulting in chronic inflammation, fibroproliferation, and angiogenesis (table 1).

Muller in 1994 [33] revealed to the atherosclerotic community that the malignant atheroma (vulnerable thin-cap fibrous atheromatous plaque) had a tendency to rupture with certain activities of daily living. He was able to relate cardiovascular events to certain triggers:

Exertion, anger, and fear are all related to a surge in adrenalin-epinephrine, norepinephrine. These vasoactive molecules when excreted would have a tendency to concentrate in areas where there are increased capillary densities such as the vulnerable, angiogenic-laden plaques as compared to the healthy segments of coronary arteries. Plaques have tendency to develop in an eccentric fashion with frequent layering of one active atheroma over another. The fixed eccentric plaque with its increased angiogenesis cannot constrict in response to this increased concentration of vasoconstrictive molecules. The vasa vasorum does communicate to the opposing side of the eccentric plaque demonstrated by Barger's work. The vasoactive molecules could stimulate the smooth muscle cells within the opposing healthy media opposite the fixed plaque and result in a slapping of the fixed diseased vessel wall. This would result in increased biomechanical stress to the unstable, vulnerable, angiogenic laden, rupture prone atherosclerotic plaque and contribute to IPH.

Recently, Stefanadis C [34] and Madjid M [35] have been able to demonstrate that the vulnerable plaque has a higher temperature than stable plaques with a temperature probe. The increased temperature of the probe was associated with elevations in C-reactive protein (CRP), serum amyloid A, and macrophage content. The authors contend that they are measuring indirectly the activity of plaque inflammation, when in fact; they may be measuring the increased temperature as a result of the increased angiogenic blood flow seen by the probe through a vulnerable and thinned fibrous cap.

To summarize, the constant injury and response to injury to the arterial vessel wall causes this natural repair and healing process (remodeling) to go awry with plaque angiogenesis and a malignant transformation (table 1).

The term intimopathy is used to describe the remodeling within the arterial vessel wall and includes ECM expansion and neovascularization of the adventitial vasa vasorum. This intimopathy is comparable to the angiogenic neovascularization seen in diabetic retinopathy and carries a similar risk when discussing IPH and retinal hemorrhage, each having a detrimental outcome. In the case of retinopathy and retinal hemorrhages there is reduced vision and blindness, whereas in intimopathy there is IPH, destabilization of the vulnerable atherosclerotic plaque, and plaque rupture with ACS and excessive remodeling of the involved plaque [10].

MS, PD, and T2DM are associated with impaired coronary collateral vessel formation via the process of arteriogenesis [10, 36].

The formation of coronary collateral vessels (arteriogenesis) is a compensatory mechanism secondary to repetitive or chronic epicardial arterial blockage by arterial vasospasm or thrombosis. The process of arteriogenesis (collateral vessel formation) begins with small (20 micron diameter vessels) preexisting, quiescent, minimally – functioning arterioles and remodels them into larger (1–2 millimeter diameter vessels) more functional arterioles capable of carrying more oxygen enriched blood to ischemic tissue (a mini auto-coronary artery bypass). The shear stress associated with a pressure gradient as a result of epicardial coronary vasospasm or thrombosis is responsible for inducing the arteriolar remodeling process. In contrast, angiogenesis begins with capillaries and ends with more capillaries (figure 6,7). Important in this remodeling process are the shared cytokines, chemokines, growth factors, the inflammatory monocyte, and the extracellular matrix metalloproteases (MMPs) (table 4) [10].

Recently Waltenberger J et al. were able to delineate the importance of the monocyte. They were able to demonstrate that monocyte migration was impaired in diabetes and associated with impaired collateral formation [37, 38]. Panutsopulos D et al. were also able to demonstrate an impairment of monocyte migration with an associated increase in basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) mRNA [39]. While the monocyte is an important cell associated with arteriogenesis and its impairment in migration will have a detrimental effect on remodeling collateralization there may also be a defect associated with MS, PD, and T2DM affecting the remodeling of the extracellular matrix (ECM).

MS, PD, and T2DM are associated with increased PAI-1 [8‐10]. PAI-1 inhibits both tissue and urokinase plasminogen activator resulting in impaired synthesis of plasmin. Plasmin is known to be very important in activating latent MMPs to active MMPs [40]. Therefore, PAI-1 excess may additionally serve to impair monocyte migration in addition to inhibiting the ECM remodeling collateralization (intima – media) of preexisting arterioles to the newly remodeled collateral arterioles (figure 7) [10].

There is a diabetes vascularization paradox in that angiogenesis is induced and arteriogenesis is impaired.

Angiogenesis and arteriogenesis are two distinct forms of postnatal vasculogenesis. Angiogenesis is induced by ischemia through the promoter Hif-1 TACGTGCT promoter and VEGF activity, whereas arteriogenesis is induced by the shear stress promotor by the shear stress responsive element (SSRE): GAGACC (figure 6) [41].

The atherosclerotic plaque angiogenic adventitial Vv within atheroscleropathy is excessive and associated with MS, PD, and overt T2DM. The Vv are induced by ischemia as the intima-media undergoes positive outward remodeling. Additionally, the Vv angiogenesis is further induced by the inflammatory process within the shoulder region of the plaque, which is associated with the known angiogenic factors: Tissue ACE, tissue factor, cytokines and growth factors (TNF alpha, VEGF, FGF). MS, PD, and T2DM are associated with multiple metabolic toxicities, which are responsible for reactive oxygen species (ROS) production (table 3). This elevated tension of redox stress contributes to the ischemia within the plaque and induces even more angiogenesis within the plaque.

In contrast arteriogenesis is induced and promoted by shear stress in normoxic conditions (table 4). Redox stress and ROS are elevated in the preexisting (un-remodeled) arteriolar endothelial milieu in MS, PD, and T2DM and is associated with endothelial dysfunction with decreased eNOS activity and decreased generation of eNO. This could result in an impaired initial vasodilatation and permeability ordinarily induced by increased shear stress in the arteriogenic process. Terjung RL et al. and Matsunaga T et al. have reported that arteriogenesis is eNOS and eNO dependent [42, 43]. Thus, the impairment in eNOS and eNO (due to redox stress and ROS) [8] could impair the initiation and progression of arteriogenesis and result in a negative effect on remodeling collateralization.

Given that collateral formation is inhibited by NOS inhibition, dysfunction or knockout, one can hypothesize that eNOS dysfunction and – or decreased eNO may decrease arteriogenesis through the following mechanism: a decrease in arteriolar vasodilation would impair the normal arteriolar vasodilation due to eNOS and eNO and decrease the pressure gradient between normal and ischemic tissue. This would impair the normal increased flow and the shear stress responsive element in the collateral vessel undergoing remodeling collateralization, as well as, decreasing endothelial cell permeability [44]. Future work is needed to establish if reversing endothelial dysfunction associated with MS, PD, and T2DM could restore improved arteriogenesis [10]. Thus it appears that a blunted endothelial NO production as noted in MS, PD, and overt T2DM could temper vascular remodeling and arteriogenesis [45].

Excess in redox stress or ROS and PAI-1 elevation could have an overall negative effect on arteriogenesis in MS, PD, and overt T2DM (table 4).

Another possibility is the existence of early advanced glycation end products (AGE) and that these AGE would contribute not only to the impaired remodeling collateralization phase but also contribute to additional collagen cross linking within the extracellular matrix. If glucotoxicity were to be better controlled this might improve the remodeling phase of arteriogenesis in T2DM by improving redox stress, endothelial dysfunction, monocyte migration, and PAI-1 levels.

MS, PD, and T2DM are associated with a diffuse endotheliopathy. This endothelial dysfunction can be related to the three vulnerable arms of the endothelial nitric oxide synthase (eNOS) reaction.

Oxidative – redox stress is elevated in MS, PD, and T2DM and results in the production of reactive oxygen species (ROS). The multiple metabolic toxicities of the A-FLIGHT acronym result in an abundance of ROS. These ROS interfere with the eNOS reaction resulting in a decrease in endothelial nitric oxide (eNO). The process of uncoupling of the eNOS reaction allows this reaction to proceed with the endothelium becoming a net producer of superoxide [O ₂ ^• ] instead of the quintessential, protective, anti-inflammatory, antioxidant eNO. When L-arginine uncouples from the eNOS enzyme via an intact necessary cofactor tetrahydrobiopterin (BH₄) the following reaction ensues:

The BH₄ requisite cofactor itself is quite sensitive to oxidative – redox stress and can be oxidized to BH₂ and BH₃, which will not run the eNOS reaction and allow uncoupling. This allows the endothelium to become a net producer of superoxide instead of eNO. As can be seen it is very important to have an adequate substrate, a proper functioning eNOS enzyme, and the necessary cofactor BH₄. Folic acid is not only a methyl donor and aids in the control of elevated homocysteine but also an electron and hydrogen donor, which brings BH₂ and BH₃ back to the requisite completely reduced BH₄. These findings would lead one to not only control the multiple metabolic toxicities (A-FLIGHT table 3) but also provide folic acid in adequate amounts to allow BH₄ to run the eNOS reaction fully re-coupled to once again become a net producer of eNO [8, 9].