Immunohistochemical Interplay in Associated Large Vessel Vasculitis and consequent Ascending Aortic Aneurysm and Review of Literature

DOINA BUTCOVAN, GRIGORE TINICA, CRISTIAN STATESCU*, DANIEL TIMOFTE*, BOGDAN CIUNTU, RADU SASCAU, RODICA RADU, LARISA ANGHEL, MIHAELA CARMINA SCHAAS*, CODRUTA BADESCU, RAOUL VASILE LUPUSORU “Grigore T. Popa” University of Medicine and Pharmacy, Department of Morpho-Functional Sciences, 16 Universitatii Str., 700115, Iasi, Romania “Grigore T. Popa” University of Medicine and Pharmacy, Department of Surgery General Surgery, 16 Universitatii Str., 700115, Iasi, Romania 3 Institute of Cardiovascular Diseases, “Grigore T. Popa” University of Medicine and Pharmacy of Iasi, 16 Universitatii Str., 700115,Iasi, Romania “Grigore T. Popa” University of Medicine and Pharmacy, Department of Ginecology, 16 Universitatii Str., 700115,Iasi, Romania “Grigore T. Popa” University of Medicine and Pharmacy, Department of Internal Medicine, 16 Universitatii Str., 700115, Iasi, Romania

to Mycobacterium tuberculosis [8]. In addition it might be initiated in the adventitia, with inflammatory cells entering through the vasa vasorum and subsequently infiltrating into all layers of the aortic wall [9]. We aimed to analyze and compare immunohistochemical (IMH) of two cases, in order to better understanding these pathologies.

Experimental part Material and methods
The study was approved by our institutional Committee of Ethics. Written informed consent was obtained from the patients.
The two cases of TA and GCA patients were one female under <50 years old and one male over > 50 years old, respectively. The study material, the residual histological blocks, were used for histological and IMH comparative examination. These cases were selected from 10 cases of AAA associated with aortitis, both having at least 3 positive criteria of diagnosis according with ACR aortitis classification.
Histologically, we analyzed 10 histological fields at a magnification of x200, for each case. We measured the wall thickness of the aortic wall. The measurements were expressed in mean values.
We analyzed immunohistochemically 10 histological sections at a magnification of x200 for each case. The degree of total wall inflammation was evaluated by relating the average inflammatory cell number to the entire studied histological section area. Data were expressed as mean values.
For the evaluation of each inflammatory cell component of the infiltrate we calculated the proportion of each cell related to total inflammatory cell number in the same histological section area. The results were expressed as percentage.
The degree of macrophages, B and T cells was morphologically classified by inflammatory cell distribution in the aortic tissue, as follows: grade 0 (<10%), grade 1 (between 10-50%), grade 2 (between 50-75%), and grade 3 (over > 75%). The end inflammatory score was the sum of the score for each type of inflammatory cell. Finally, we compared the score results.

Statistical analysis
The IMH comparison of composition and distribution of the inflammatory infiltrate of the AAA wall between GCA and TA case was assessed. Data are expressed as mean values and percentages, calculated using Excel software (Microsoft Corporation, Redmond, WA, USA).

Results and discussions
The average values of aortic thickness (intima, media, and adventitia) in the GCA and TA are presented in Table 1. The aortic wall thickness was about double in TA (4034 µm) comparative with GCA (2122 µm). The medial layer of GCA was thicker than that of TA, while the adventitia was thinner, respectively.
Histologically, in GCA we found a fibrous extracellular matrix intima containing foci of mononuclear inflammatory cells located mainly at I-M junction, medial fibrous areas and nodular and band-like inflammatory foci in association with neovascularization, and adventitial nodular and band-like inflammatory foci and intense neovascularization (Fig. 1a).
In TA, we noted a thickened intima by collagenous bands disposed parallel to the endothelium, medial areas of elastic lamina destruction and nodular and band-like inflammatory foci in association with neovascularization, and thickened adventitia by connective extracellular matrix and neovascularization (Fig. 1b). Distribution of inflammatory cells within aortic layers, in giant cell arteritis (GCA) and Takayasu's arteritis (TA) are showed in Table 1.

Table 1 IMMUNOHISTOCHEMICAL ASSESSMENT OF INFLAMMATION IN GIANT CELL ARTERITIS (GCA) AND TAKAYASU'S ARTERITIS (TA)
In the aortic wall, inflammatory cells are of 1.16 times more in GCA (1117) than in TA (958), and with different values in the layers of the aortic wall. Inflammatory infiltrate is of 11.6 higher in the intima of the aorta in GCA (221) than in TA (19) and only of 1.79 times higher in aortic adventitia in GCA (620) than TA (345).
Inflammatory cell ratio was approximately double (2.15) in media of the aortic wall in TA (594) than GCA (276) or less than half times less (0.46) in GCA than in TA.
So, our results showed that both aortitis are panarteritis, with similar histology and different distribution and degrees of inflammation within aortic all. In our opinion, the inflammation in both aortitis is a chronic phase with still persistent inflammation in TA.
The proportion of inflammatory cells within aortic wall layers is showed in Table 2.  Comparative layer inflammatory distribution in GCA vs TA was the following: -In GCA, intima contains 221 mononuclear inflammatory cells, of which 21% are T -lymphocytes, 8.5% are Blymphocytes and 70.5% are macrophages while in TA, all 19 inflammatory cells were T -lymphocytes located only in the intima.
-The medial layer contains in GCA 276 mononuclear inflammatory cells, of which 39.2% are T -lymphocytes, 8.6% are B -lymphocytes and 52.2% are macrophages, while in TA of 594 mononuclear inflammatory cells, 60.2% are Tlymphocytes, 28.2% are B -lymphocytes and 11.6% are macrophages.
Entire aortic wall inflammatory score in GCA and TA is showed in Table 3. Total wall inflammatory infiltrate score was bigger in TA (4) than in GCA (3).
Individual inflammatory cell score in GCA and TA is showed in Table 4.

Table 4 INFLAMMATORY CELL SCORE IN GIANT CELL ARTERITIS (GCA) AND TAKAYASU'S ARTERITIS (TA) WITHIN AORTIC WALL LAYERS
The inflammatory infiltrate had a similar distribution in the aortic all, with different proportion of the inflammatory cells (Fig. 2.). Fig. 2. Giant cell arteritis:a-adventitial CD3; b-medial CD3; c-medio-adventitial CD68; d-adventitial CD20. Takayasu's arteritis: e-intimo-medial CD3;f-medio-adventitial CD68; g-adventitial CD20 Comparative inflammatory score in GCA and TA was assessed according with two criteria. Depending on the aortic wall involved layers, the total inflammatory score in GCA was 9, the same in intima (3), media (3) and adventitia (3), while the total inflammatory score in TA was 8, having 0 value in intima and 4, in both media and adventitia. Depending on the type of inflammatory cells in the entire wall thickness, the inflammatory score in GCA was 3 for Tlymphocytes (CD3), 1 for B -lymphocytes (CD20) and 5 for macrophages (CD68), while the inflammatory score in TA was 4 for Tlymphocytes, 2 for B -lymphocytes and 2 for macrophages.
In GCA, the proportion of the inflammatory cells of 3:1:5 probably denotes a chronic healing process with a mild recent activation by an exogenous agent, maybe viral one. In TA, the proportion of the inflammatory cells of 4:2:2 probably denotes a persistent chronic inflammation which is still active (due to persistence of antigen stimulation related by tuberculosis history), demonstrated by extensive destructive areas. Both aortitis have about the same inflammatory score, but they are in various evolutionary stages related to different intensity of inflammatory activity.
Takayasu arteritis and GCA are the two main variants of LVV. Takayasu's arteritis is a LVV affecting elastic arteries such as the aorta and its branches, while GCA refers to involvement of the aorta and its proximal or extracranial aortic branches. In a patient with GCA, Prieto-Gonzalez showed the aortic involvement using computed tomography angiography (angio-CT) and aortic biopsy [10].
These two conditions have been considered separate entities because of the observed differences in the age at onset, showing clinical features, geographic distribution and location of arterial involvement (8). Recently, Polachek A et al have proposed that TA and GCA may exist within a clinical spectrum of a single disease [11].
Both our cases had AAA and aortic insufficiency at hospital presentation, having symptoms related to this vascular disease. Both LVV cases had positive inflammatory markers and angiographic signs of ascending aortic involvement, completed with specific signs of aortic branch stenosis in TA case. The artery biopsy was the gold standard for diagnosis of these aortitis [12].
According with Eberhardt RT (13) et al, we also found at GCA histology exam a chronic evolutionary stage of the GCA characterized by moderate intimal thickening by fibromyxoid changes, medial granulomas with rare multinucleated giant cells and medial fibrosis, and adventitial fibrosis. The inflammation showed to be marked in the adventitia, too [13].
As Vaideeswar P notes, we also reported at histological TA exam a chronic evolutionary stage, consisting in thicker, collagenous intima, medial focal granulomas related to elastic fibers fragmentations and medial fibrosis, and focal adventiceal perivasa vasorum inflammatory infiltrate. In addition, we also noted neovascularization within the entire wall [14].
Due to similar GCA and TA histopathological findings, the biopsy results only may not differentiate between these two vasculitis. So, in discrimination of the two aortitis, the age in both and CT angiography in TA are essential tools.
Similar with Chakravarti R appreciation, we consider that the more severe medial inflammation leads to loss of smooth muscle cells and elastic fibers, medial weakening and aneurysm formation. In addition, we revealed a significant adventitial reactive fibrosis in the intima, media and adventitia and neovascularization at the medio-adventitial junction in TA [15].
In both TA and GCA we found parietal inflammatory lesions produced in the adventitia through the vasa vasorum, allowing the lymphocytes to gain access to the arterial wall, as Noris M. also observed [16].
Cell-mediated autoimmunity has been clearly involved in the physiopathology of vascular cell injury in TA and GCA, due to the highest values of T -lymphocytes and macrophages in these two diseases. We did not found GCA associated infections (a virus infection cannot be excluded) while we noted a history of tuberculosis infection in TA patient.
Furthermore, an increased immune response to Mycobacterium tuberculosis has been reported by Moraes MF in TA cases, too (8). We consider, that using lymphocytic and histiocytic markers (CD68) in suspected GCA and TA, we can detect residual arteritis in patients with resolving disease.
If humoral immunity takes part in the physiopathology of GCA and TA is not clear. We found more B Lys in GCA than TA, and we consider that both aortitis have a persistent active inflammation of different degrees.
LVV adventitial analysis showed that inflammation was most prominent in GCA, with infiltration of B and T cells. In TA, we found adventitial inflammatory foci, with B and T cells surrounding vasa vasorum. The distribution of immunostainings seen at the outer media border indicates that the majority of the inflammatory cells enters the arterial wall from adventitial microvessels, migrate through the media and the inflammatory activity focuses finally on the media and intima.

Conclusions
This study showed that beyond the role of T cells in GCA and TA, B cells are also involved in humoral responses in patients with activated disease. The initial site of inflammation seems to be around the adventitial vasa vasora which is an early stage in the development of classic transmural inflammatory infiltration. The medial inflammatory areas related to prominent elastic fibers fragmentation was the cause of medial weakening and aneurysm formation.