**5. Metalloproteinases and aneurysms**

66 Aneurysm

non-matrix proteins which can be substrates for various MMPs. Metalloproteinases are involved in the activation of latent forms of effective proteins. For example, MMP-2, MMP-3 and MMP-9 can activate interleukin 1 (IL-1). They can also act on active cytokines, IL-1 undergoes subsequent degradation catalyzed by MMP-3. Metalloproteinases can alter cell

Metalloproteinases are not indiscriminately released by cells. They are secreted to or anchored to cell membrane. MT-MMPs have a specific transmembrane domain placing them in a certain position. Other metalloproteinases can be bound by specific cell-MMP interactions. This phenomenon allows an exact localization of their proteolytic activity [1,2].

Metalloproteinases are encoded as inactive proenzymes, zymogens. They undergo proteolytic activation. This process can take place either intracellulary or extracellulary. One third of MMPs are activated by intracellular serin protease, furin. This process takes place in trans-Golgi network. A number of MMPs has a cleavage site for other metalloproteinases. MMP-3 activates proMMP-1 and pro-MMP-7. Some metalloproteinases have been described

*In vivo* studies indicate that reactive oxygen species (ROS) generated by neutrophils can both activate and subsequently inactivate MMPs. Hypochlorus acid (HClO) generated by neutrophil myeloperoxidase and hydroxyl radicals can activate proMMP-1, proMMP-7 and proMMP-9, whereas peroxynitrate can activate proenzymes of MMP-1, MMP-2 and MMP-9. This process enables a control of burst of proteolytic activity within an inflammatory setting. Like some other proteases, activity of MMPs is controlled also by two other mechanisms, regulation of gene expression and specific inhibitors. MMP-2 is constitutively expressed and regulation of its activity occurs by either activation or inhibition. Expression of a number of metalloproteinases is up-regulated during various pathological conditions. Among them inflammation is the most studied setting. MMPs are inhibited by α-2 macroglobulin and tissue inhibitors of metalloproteinases (TIMPs). There are four TIMPs. Their secretion is also regulated and represents another point in a network of control of the activity of metalloproteinases. TIMP-3 is primarily bond to ECM and allows a regulation of MMPs' activity in the very site of their action. The network of the control of the activity of metalloproteinases is complex and very precise. Sometimes TIMP interacts with proMMP and inactivate other MMP, e.g. a complex of TIMP-1 and proMMP-9 inactivates MMP-3.

Protection from MMP degradation represents the next step in this sophisticated network of diverse interactions. Neutrophil gelatinase-associated lipocalin (NGAL) bounds to MMP-9

Metalloproteinases can be detected in all three layers of a vascular wall. Endothelium can produce MMP-1 and MMP-2. Smooth muscle cells (SMC) of both intima and media are the

protecting this metalloproteinase from its degradation [1,2].

**4. Localisation of metalloproteinases in a vascular wall** 

surface proteins such as receptors and act on microbial peptides.

**3. Activation of metalloproteinases** 

to be activated by kallikrein or plasmin.

The most studied aneurysm is abdominal aortic aneurysm (AAA), far less research has been focused on aneurysms of cerebral arteries and thoracic aortic aneurysm. All aneurysms are characterized by the destruction of the structural integrity of the extracellular matrix proteins, mainly collagens and elastin. MMPs involved in this pathology can origin both from the cells that physiologically constitute the arterial wall and are stimulated to secrete MMPs, i.e. endothelium, SMC and cells that infiltrate the arterial wall in a response to various stimuli [1, 3, 6-9].

Many scientists points out that cells constituting an inflammatory infiltrate are the major source of metalloproteinases involved in the development of aneurysms. Studies of samples derived from patients undergoing surgery for AAA demonstrated that macropgages from the inflammatory infiltrate can express MMP-1, MMP-2, MMP-3, MMP-9 and MT-1MMP. Metalloproteinase-2 was often detected in cells physiologically constituting the arterial wall, but was absent in macrophages within aneurysms. The pathogenesis of AAA and aneurysms of cerebral arteries differs as these vessels present different types of arteries and there are some differences in the physical characteristic of blood flow in them. Recent experimental studies carried on animals confirmed the role of macrophage infiltration in the formation of intracranial aneurysms. A degranulation of mast cells induces the expression and activation of MMP-2 and MMP-9. Inhibitors of mast cell degranulation inhibited the development of cerebral aneurysms in experimental rats [10, 11].

Human studies confirmed that the expression of metalloproteinases within the AAA is greater than in other sites, remote from the dissection. Nishimura *et al.* observed a different profile of MMP activation in small size abdominal aortic aneurysms, less than 45 mm and large size AAA with diameter exceeding 45 mm. In small size AAA MMP-2 and MMP-9 presented greater gene expression whereas in large size AAA membrane type-1 metalloproteinase and MMP-9 had greater expression. The same study demonstrated also differences in the distribution of the metalloproteinases in the arterial wall. MMP-2 was detected mainly in the intima, whereas MMP-9 was present both in intima and adventitia.

Nishimura *et al.* also observed a significant correlation of the expression of MMP-2 and MMP-9 and between each of these metalloproteinases and TIMP-1 [6].

The degeneration of collagen and elastin leading to the development of aneurysms is a multifactorial process. Various factors may take part in the stimulation of both cells constituting the arterial wall and cells infiltrating it to produce MMPs. Aortic wall is subjected to cyclic stretching because of pulsative blood flow which is a normal physiological condition. AAA is often accompanied by an intraluminal thrombus. It causes that some cells within the aneurysm may be subjected to hypoxia. Experimental study of Oya *et al*. revealed that macrophages cultured in conditions subjected to cyclic stretching under normoxia and hypoxia which simulated the pulsative blood flow and hypoxia due to thrombus presented an increased MMP-9 production. These macrophages produced interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) leading to increased apoptosis of vascular smooth muscle cells. Hypoxia was also demonstrated to augment the expression of MMP-1, MT-1 MMP, MMP-2, MMP-7 and MMP-9 in SMC derived from human aorta [12, 13].

Nowadays many scientists focus their research on finding new factors which may augment the secretion of metalloproteinases by the cells present in aneurysms. Experimental studies confirmed that stenosis resulting in a turbulent blood flow can be the next factor increasing expression of MMP-2 and MMP-9 within abdominal aortic aneurysm. Interesting results were obtained by Stolle *et al*. Mice exposed to cigarette smoke and angiotensin II treatment had increased the incidence of AAA and higher gene expression of MMP-2, MMP-3, MMP-8, MMP-9 and MMP-12 in aorta and increased proteolytic activity of two most investigated metalloproteinaes, MMP-2 and MMP-9. Although each of this two factors alone induced minor changes, their combination accelerated the pathologic process. Exposure of the arterial wall to an increased concentration of angiotensin II represents conditions that may be observed in patients with arterial hypertension. This experiment demonstrates that coexistance of arterial hypertension and smoking augments the risk of a development of aneurysm [14, 15].

Studies of Zhang *et al*. demonstrated that human AAA tissues had elevated levels of advanced glycation end products (AGEs) and their receptor (RAGE). In experimental model this group of researchers observed that AGEs induce the production of MMP-9 by macrophages. An increased serum concentration of AGEs accompanies poorly controlled diabetes mellitus. These results indicate that such patients may be at a greater risk of a development of AAA [16].

Abdominal aortic aneurysm is characterized not only by the destruction of its structural integrity of the extracellular matrix protein and inflammatory infiltrate but also by intensive neovascularisation. These new blood vessels developing inside the arterial wall in a place of growing aneurysm are the next source of metalloproteinases degrading ECM. Immature neovessels express MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9 and MMP-12 [8].

Polish scientists have observed that the intraluminal thrombus occurring within AAA may modulate the activity of MMP-8, MMP-9, neutrophil elastase and TIMP-1. Specimens from patients with thin, less than 10 mm, thrombus-covered wall of AAA presented significantly higher activity of 3 evaluated metalloproteinases and lower TIMP-1 concentration than thick, exceeding 25 mm, thrombus-covered wall. The intraluminal thrombus may exert its pathological effect through trapping erythrocyte and neutrophils and monocytes. The exact mechanism of activation of MMP in these conditions has not been fully elucidated. Scientists consider various factors activating metalloproteinases, such as hypoxia caused by reduced blood flow or oxidative stress in trapped blood cells. This aspect needs further evaluation [17].

Japanese researchers compared the activity of MMP-2 and MMP-9 in ruptured and unruptured middle cerebral artery dissections obtained during neurosurgery in the same patient. Both metalloproteinases presented greater expression in the ruptured dissection [9].
