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Objective: In the present study, we used a two-kidney-two-clip (2k2c) stroke-prone renovascular hypertension rat model (RHRSP) to investigate the protective effects of ligustrazine (TMP) on cerebral arteries and to examine PI3K/Akt pathway behavior under this protection., Methods: The cerebral artery remodeling was induced by 2k2c-induced renovascular hypertension. Brain basilar artery tissues were isolated and their histological changes were detected through H&E and EVG staining, α-SMA IHC staining, and transmission electron microscopy at four, eight, and twelve weeks after 2k2c surgery, both with and without TMP treatment. Meanwhile, the ET-1, Ang II, and NO levels in basilar arteries and plasma were determined. Furthermore, the PTEN expression and the activation of PI3K/Akt in basilar artery tissues were detected through IHC and Western Blot. In addition, the primary basilar artery smooth muscle cells (BASMCs) were cultured and TMP protection of BASMCs stimulated with ET-1/Ang II in the presence or absence of insulin-like growth factor 1 (IGF-1) was determined., Results: TMP attenuated basilar artery remodeling, decreased ET-1 and Ang II levels and increased NO level in basilar arteries and plasma of RHRSP rats. Moreover, TMP reduced BASMCs proliferation upon ET-1/Ang II stimulation. We also found that TMP could effectively suppress the activation of PI3K/Akt in 2k2c-RHRSP rat basilar artery and ET-1/Ang II stimulated BASMCs. Most importantly, IGF-1, as an activator of PI3K/Akt, could damage the protective effect of TMP., Conclusions: TMP exerts its protective effects and prevents basilar artery remodeling in RHRSP rats at least partly through the inhibition of PI3K/Akt pathway., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Rationale: Giant cell arteritis (GCA)-a primary vasculitis of medium and large arteries-is associated with vessel wall damage, elastic membrane fragmentation, and vascular remodeling. Proteinases are believed to contribute to pathogenesis by degrading extracellular matrix and causing tissue injury., Objective: The MMP (matrix metalloproteinase)-9-a type IV collagenase-is produced in the vasculitic lesions of GCA. It is unknown which pathogenic processes are MMP-9 dependent., Methods and Results: The tissue transcriptome of GCA-affected temporal arteries contained high amounts of MMP-9 transcripts, and immunostaining for pro-MMP-9 localized the enzyme to wall-infiltrating macrophages. MMP-2 and MMP-9 transcripts were also abundant in monocytes and monocyte-derived macrophages from patients with GCA. Patient-derived monocytes outperformed healthy monocytes in passing through engineered basement membranes. GCA CD (cluster of differentiation) 4 + T cells required MMP-9-producing monocytes to penetrate through matrix built from type IV collagen. In vivo functions of MMP-9 were tested in a human artery-SCID (severe combined immunodeficiency) chimera model by blocking enzyme activity with a highly specific monoclonal antibody or by injecting rMMP-9 (recombinant MMP-9). Inhibiting MMP-9 activity profoundly suppressed vascular injury, decreased the density of inflammatory infiltrates ( P<0.001), reduced intramural neoangiogenesis ( P<0.001), and prevented intimal layer hyperplasia ( P<0.001). rMMP-9 amplified all domains of vasculitic activity, promoted assembly of T-cell infiltrates ( P<0.05), intensified formation of new microvessels ( P<0.001), and worsened intimal thickening ( P<0.001). Systemic delivery of N-acetyl-proline-glycine-proline-a matrikine produced by MMP-9-mediated gelatinolysis-had limited vasculitogenic effects., Conclusions: In large vessel vasculitis, MMP-9 controls the access of monocytes and T cells to the vascular wall. T cells depend on MMP-9-producing monocytes to pass through collagen IV-containing basement membrane. Invasion of vasculitogenic T cells and monocytes, formation of neoangiogenic networks, and neointimal growth all require the enzymatic activity of MMP-9, identifying this protease as a potential therapeutic target to restore the immunoprivilege of the arterial wall in large vessel vasculitis.

Background: Giant cell arteritis, a chronic autoimmune disease of the aorta and its large branches, is complicated by aneurysm formation, dissection, and arterial occlusions. Arterial wall dendritic cells attract CD4 + T cells and macrophages to form prototypic granulomatous infiltrates. Vasculitic lesions contain a diverse array of effector T cells that persist despite corticosteroid therapy and sustain chronic, smoldering vasculitis. Transmural inflammation induces microvascular neoangiogenesis and results in lumen-occlusive intimal hyperplasia. We have examined whether persistent vessel wall inflammation is maintained by lesional T cells, including the newly identified tissue-resident memory T cells, and whether such T cells are sensitive to the cytokine-signaling inhibitor tofacitinib, a Janus kinase (JAK) inhibitor targeting JAK3 and JAK1., Methods: Vascular inflammation was induced in human arteries engrafted into immunodeficient mice that were reconstituted with T cells and monocytes from patients with giant cell arteritis. Mice carrying inflamed human arteries were treated with tofacitinib or vehicle. Vasculitic arteries were examined for gene expression (reverse transcription polymerase chain reaction), protein expression (immunohistochemistry), and infiltrating cell populations (flow cytometry)., Results: Tofacitinib effectively suppressed innate and adaptive immunity in the vessel wall. Lesional T cells responded to tofacitinib with reduced proliferation rates (<10%) and minimal production of the effector molecules interferon-γ, interleukin-17, and interleukin-21. Tofacitinib disrupted adventitial microvascular angiogenesis, reduced outgrowth of hyperplastic intima, and minimized CD4 + CD103 + tissue-resident memory T cells., Conclusions: Cytokine signaling dependent on JAK3 and JAK1 is critically important in chronic inflammation of medium and large arteries. The JAK inhibitor tofacitinib effectively suppresses tissue-resident memory T cells and inhibits core vasculitogenic effector pathways., (© 2017 American Heart Association, Inc.)

Objectives: Gelatinases (MMP2 and MMP9) are expressed in giant-cell arteritis (GCA) and are thought to play a role in vessel disruption. However, their activation status and enzymatic activity have not been evaluated. Our aim was to investigate the distribution and proteolytic activity of gelatinases in GCA lesions at different stages., Methods: Expression of MMP2, MMP9, MMP2-activator MMP14 and their natural inhibitors TIMP1 and TIMP2 was determined by real-time PCR and immunohistochemistry in temporal artery sections from 46 patients and 12 controls. MMP activation status and enzymatic activity were assessed by gelatin and film in situ zymography., Results: Vascular smooth muscle cells from normal specimens constitutively expressed pro-MMP2 and its inhibitor TIMP2 with no resulting proteolytic activity. In GCA MMP2, MMP9 and MMP14 were strongly expressed in their active form by infiltrating leucocytes. Inflamed arteries also expressed TIMP1 and TIMP2. However, the MMP9/TIMP1 and MMP2/TIMP2 ratios were higher in patients compared with controls, indicating an increased proteolytic balance in GCA which was confirmed by in situ zymography. Maximal gelatinase expression and activity occurred at the granulomatous areas surrounding the internal elastic lamina (IEL). Myointimal cells also expressed MMPs and exhibited proteolytic activity, suggesting a role for gelatinases in vascular remodelling and repair., Conclusions: GCA lesions show intense expression of gelatinases. Activators and inhibitors are regulated to yield enhanced gelatinase activation and proteolytic activity. Distribution of expression and proteolytic activity suggests that gelatinases have a major role not only in the progression of inflammatory infiltrates and vessel destruction but also in vessel repair.

Background: Both matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) have been postulated to play roles in the pathophysiology of giant cell arteritis (GCA) because of their ability to degrade elastin. Understanding the specific mediators of arterial damage in GCA could lead to new therapeutic targets in this disease., Methods and Results: Temporal artery biopsy specimens were obtained from 147 consecutive patients suspected of GCA. Clinical and histopathological data were collected according to protocol. Using immunohistochemistry, we compared the expression of MMP-2 and MMP-9 in the temporal artery biopsies of both GCA cases (n=50) and controls (n=97). MMP-9 was found more frequently in positive than in negative temporal artery biopsies (adjusted odds ratio [OR], 3.20; P=0.01). In contrast, the frequency of MMP-2 was not significantly different between positive and negative biopsies (adjusted OR, 2.18; P=0.22). Both MMP-2 and MMP-9 were found in macrophages and giant cells near the internal elastic lamina and in smooth muscle cells and myofibroblasts of the media and intima. MMP-9 was also found in the vasa vasorum. MMP-9 but not MMP-2 was associated with internal elastic lamina degeneration, intimal hyperplasia, and luminal narrowing, even after adjustment for possible confounding variables., Conclusions: MMP-9 appears more likely than MMP-2 to be involved in the pathophysiology of GCA. MMP-9 not only participates in the degradation of elastic tissue but also is associated with intimal hyperplasia, subsequent luminal narrowing, and neoangiogenesis. The expression of MMP by smooth muscle cells implicates these cells as potential secretory cells in GCA.

Background: The incidence of cardiac allograft vasculopathy increases with time after heart transplantation. Allograft vasculopathy is associated with endothelial dysfunction and reduced endothelium-dependent nitric oxide-mediated vascular effects. In this study, temporal changes in endothelial nitric oxide synthase (NOS3) expression in human myocardial biopsies were investigated during the first 3 years after heart transplantation., Methods: In each patient (n = 7), the immunohistochemical presence of NOS3 and inducible nitric oxide synthase were examined in serial biopsies taken at 1, 4, and 26 weeks and at 1, 2, and 3 years after transplantation., Results: Endothelial nitric oxide synthase was present in vascular endothelial cells in all biopsies at the time of transplantation. A rapid fall within the first months in the number of NOS3-positive biopsies was observed, with a possible difference in the rate of disappearance among the capillaries, the arterial endothelium, and the venous endothelium. After 2 years, very little NOS3 could be detected. Inducible nitric oxide synthase was present in vascular smooth muscle cells throughout the study period and did not change., Conclusion: Endothelial nitric oxide synthase immunoreactivity is gradually lost after heart transplantation. These changes may be responsible for the coronary endothelial dysfunction often seen after human cardiac transplantation.

Giant cell arteritis (GCA) is a systemic vasculitis preferentially affecting large and medium-sized arteries. Inflammatory infiltrates in the arterial wall induce luminal occlusion with subsequent ischemia and degradation of the elastic membranes, allowing aneurysm formation. To identify pathways relevant to the disease process, differential display-PCR was used. The enzyme aldose reductase (AR), which is implicated in the regulation of tissue osmolarity, was found to be upregulated in the arteritic lesions. Upregulated AR expression was limited to areas of tissue destruction in inflamed arteries, where it was detected in T cells, macrophages, and smooth muscle cells. The production of AR was highly correlated with the presence of 4-hydroxynonenal (HNE), a toxic aldehyde and downstream product of lipid peroxidation. In vitro exposure of mononuclear cells to HNE was sufficient to induce AR production. The in vivo relationship of AR and HNE was explored by treating human GCA temporal artery-severe combined immunodeficiency (SCID) mouse chimeras with the AR inhibitors Sorbinil and Zopolrestat. Inhibition of AR increased HNE adducts twofold and the number of apoptotic cells in the arterial wall threefold. These data demonstrate that AR has a tissue-protective function by preventing damage from lipid peroxidation. We propose that AR is an oxidative defense mechanism able to neutralize the toxic effects of lipid peroxidation and has a role in limiting the arterial wall injury mediated by reactive oxygen species.

Giant cell arteritis (GCA) is a relatively common granulomatous arteritis of unknown etiology which mainly occurs in elderly people. Using histopathological findings from-seven biopsy cases of temporal artery and one autopsy case of GCA, and performing immunocytochemical staining for matrix metalloproteinase (MMP)-2 and -9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 and -2, we tested the hypothesis that an imbalance between MMPs and TIMPs may be a critical determinant in developing severe intimal hyperplasia and luminal stenosis. All biopsy cases revealed nearly complete luminal occlusion of the temporal artery with active lymphocytic infiltrate, fragmentation of internal lamina and median elastic fibers. Four of seven cases revealed typical GCA. The autopsy case was systematically sampled for histological examination, revealing GCA in the ascending aorta, main branches of aorta and coronary artery. Immunocytochemical staining revealed intense staining for MMP-2 and -9 in fragmented media of the aorta and artery, and less positive staining for TIMP-1 and -2 at the MMP-positive media. In situ hybridization revealed intense positive staining for TIMPs in GCA despite weak immunocytochemical staining for TIMPs. Control cases were negative for TIMPs by immunocytochemical staining whereas RNA message level was mildly positive at a lesser intensity than that of GCA. Granulomatous tissue of fibroblasts and giant cells were most intensely positive for MMPs. The presence of markedly increased MMPs and less increased TIMPs in GCA may implicate an MMPs-TIMPs imbalance in the pathogenesis of GCA.

Objective: alpha 1-Antitrypsin (AAT) and alpha 2-macroglobulin (AMG) are elastase inhibitors that bind the enzyme and reduce measured levels of free elastase. It was recently demonstrated that some patients with intracranial aneurysms have significantly elevated plasma elastase (PE) levels. Although this elevation is unrelated to plasma AAT, it is unknown whether abnormal AAT phenotypes or reduced AMG levels play a role. Moreover, the pathological significance of this elevation is not understood., Methods: Plasma from 24 patients with aneurysms (ruptured, n = 15; unruptured, n = 9) and 10 age-matched patients who comprised a control group was analyzed for PE and AMG levels by enzyme-linked immunosorbent assay and for AAT phenotype by isoelectric focusing. Sections of superficial temporal temporal artery obtained from these patients at the time of surgery were examined for evidence of elastin degradation by using a van Gieson stain, with scoring on a nine-point quantitative scale., Results: Patients with aneurysms showed significantly elevated PE levels (119 +/- 28 versus 17 +/- 7 micrograms/ml, P < 0.05), but AMG levels were not decreased. AAT phenotypic abnormalities were observed in 10% (2 of 20) of the patients with aneurysms, but this was not different from the expected population incidence (7%). Elastin degradation scores were significantly higher in patients with aneurysms than in patients control group (4.26 +/- 0.54 versus 1.21 +/- 0.43, P < 0.05). In addition, patients with higher elastase levels (> 80 micrograms/ml) demonstrated 55% higher degradation scores than did those with lower elastase levels (< 80 micrograms/ml)., Conclusion: These data suggest that high PE levels may play a role in systemic arterial elastin degradation seen in patients with intracranial aneurysms. These data also support the contention that elevated elastase levels are not the result of decreased protease inhibitor levels. Although PE levels were significantly higher for the entire group of patients with aneurysms, this assay has relatively low sensitivity for predicting the presence of unruptured aneurysms. Additional study is necessary to determine whether serum elastase levels greater than 80 micrograms/ml, in the setting of other risk factors, are useful in identifying asymptomatic patients for additional screening.

Inflammation precedes erosion and rupture of atherosclerotic atheromas and aneurysms. Inflammatory infiltrates of macrophages have been shown to secrete proteolytic enzymes, including matrix metalloproteinases (MMPs), that weaken the arterial wall. The effect of inflammation on arterial structure and remodeling can be studied in primary vascular inflammatory diseases such as in temporal arteritis. We examined the 72-kd gelatinase (MMP-2) and the 92-kd gelatinase (MMP-9) in inflamed and uninvolved temporal arteries from 10 patients with temporal arteritis and 5 controls by immunohistochemistry. The substrates of these enzymes, type IV collagen and elastin, were detected by immunohistochemistry and histochemical staining, respectively. Both diseased and normal artery specimens had moderate staining for immunoreactive MMP-2. Temporal arteritis specimens had clearly enhanced immunostaining for MMP-9 compared with normal arteries. MMP-9 was specifically localized to macrophages in regions of internal elastic lamina disruption, which may thus be of pathological significance.

An electron microscopic immunocytochemical study was undertaken to clarify ultrastructural localization and translocation of nitric oxide synthase (NOS) in endothelial cells (EC) of the human cerebral and superficial temporal arteries (STA) employing antibody against endothelial NOS (EC-NOS). NOS immunoreactivity was found in all EC examined, in association with the plasma membrane and cytoplasmic organelles such as endoplasmic reticulum, Weibel-Palade body and subplasmalemmal vesicles, and in the cytoplasm devoid of organelles and extracellular regions, irrespective of arteries. The immunoreactivity in subplasmalemmal vesicles was, however, demonstrated only in human cerebral arteries. In the human STA exposed to bradykinin which induces EC-NOS phosphorylation, the gold particles significantly increased in the cytosol and decreased in the areas associated with cytoplasmic organelles; however, the number of particles did not change significantly in the plasma membrane. The results implicate that NOS may be translocated from the area associated with cytoplasmic organelles to cytosol following EC exposure to bradykinin.

Choline-acetyltransferase (ChAT) activity was surveyed in segments of cranial arteries--both cerebral and extracerebral--from the cat. High levels were found in pial arteries, both cerebral and cerebellar, and in the arteries to salivary glands, tongue, and nose. Intermediate levels were found in the external and internal maxillary arteries and many of their branches. Enzyme levels in the arteries supplying the head--common carotid, vertebral, and in several systemic arteries and veins and also the lingual vein--were probably not significant. Only those vessels that have higher ChAT contents show capacity for neurogenic vasodilation. The dilation of segments of a number of these arteries, the basilar, middle cerebral, lingual, and internal maxillary, is reduced significantly by atropine (5 X 10(-7) M). ChAT activity did not correlate with vessel norepinephrine content. The data may be interpreted as defining a functional vasodilator system to the head encompassing both cerebral and extracerebral arteries that depends in part on a functional cholinergic link involving a muscarinic receptor. It is separate from the adrenergic outflow. The tissues supplied by vasculature receiving this type of innervation are of ectodermal origin.