Monocytic adhesion molecule expression and monocyte-endothelial cell dysfunction are increased in patients with peripheral vascular disease versus patients with abdominal aortic aneurysms.

BACKGROUND: Statin therapy is used in the medical management of patients with peripheral vascular disease (PVD) and abdominal aortic aneurysm (AAA) for the pleiotropic and anti-inflammatory benefits. We hypothesize that the inflammatory mechanisms of monocyte-endothelial cell interactions in endothelial barrier dysfunction are more significant in patients with PVD compared with those with AAA. The purpose of this study was to assess patient peripheral blood monocyte adhesion molecules by flow cytometry and monocyte-induced endothelial barrier dysfunction by using an in vitro endothelial cell layer and electric cell-substrate impedance sensing (ECIS) system. METHODS: Peripheral blood was collected from patients with either PVD (ankle-brachial index <0.9, toe-arm index <0.8, or required lower extremity vascular intervention) or AAA (aortic diameter >3.0 cm). Monocytes were isolated from fresh whole blood using an accuspin-histopaque technique. The separated monocytes underwent flow cytometry analysis to evaluate the expression levels of the cell membrane adhesion molecules: CD18, CD11a/b/c, and very late antigen-4. Endothelial cell function was assessed by adding monocytes to an endothelial monolayer on ECIS arrays and coculturing overnight. Peak changes in transendothelial electrical resistance were measured and compared between patient groups. RESULTS: Twenty-eight monocyte samples were analyzed for adhesion molecules (PVD, 19 and AAA, 9) via flow cytometry, and 11 patients were evaluated for endothelial dysfunction (PVD, 7 and AAA, 4) via ECIS. There was no significant difference between risk factors among PVD and AAA patients except for age, where AAA patients were significantly older than PVD patients in both flow cytometry and ECIS groups (P=0.02 and 0.01, respectively). There were significantly higher levels of adhesion molecules CD11a, CD18, and CD11c (averaged mean fluorescent intensity P values: 0.047, 0.038, and 0.014, respectively) in PVD patients compared with AAA patients. No significant difference was found for CD11b and very late antigen-4 expression (P=0.21 and 0.15, respectively). There was significantly more monocyte-endothelial cell dysfunction in patients with PVD versus patients with AAA, with a maximal effect seen at 15h after monocyte addition (P=0.032). CONCLUSIONS: Patients with PVD have increased expression levels of certain monocyte adhesion molecules and greater monocyte-induced endothelial layer dysfunction compared with those with AAA. This may lead to other methods of targeted therapy to improve outcomes of these vascular patients.

Serum metalloproteinases MMP-2, MMP-9, and metalloproteinase tissue inhibitors in patients are associated with arteriovenous fistula maturation.

OBJECTIVE: Many vascular surgeons construct arteriovenous fistulas (AVFs) for hemodialysis access as the primary choice access. A significant number of AVFs fail to mature, however, leading to patient frustration and repeated operations. Metalloproteinase (MMP) activity, particularly MMP-2 and MMP-9, may be important for AVF maturation. We therefore sought to identify whether serum MMP levels could serve as a biomarker for predicting future successful AVF maturation. METHODS: Blood was collected from patients with chronic renal insufficiency at the time of surgery for long-term hemodialysis access. Serum was separated from whole blood and ultracentrifuged at 1000g for 10 minutes. Serum aliquots were frozen at -80°C until used for analysis. Enzyme-linked immunosorbent assay was used to assay levels of MMP-2, MMP-9, and tissue inhibitor of metalloproteinase type 2 (TIMP-2), and TIMP type 4 (TIMP-4). Clinical end points were used to divide patients into failed and matured AVF groups. Successful maturation was considered in patients who had specific duplex findings or 1 month of successful two-needle cannulation hemodialysis. MMP/TIMP ratios were calculated as an index of the MMP axis activity because MMP activity parallels alterations in TIMP levels. RESULTS: Of 20 enrolled patients, AVF maturation was successful in 13 and failed in 7. Serum levels of MMP-2/TIMP-2 were significantly higher in patients with matured AVFs vs levels in those that failed (P = .003). Similarly, a trend toward increased serum levels of MMP-9/TIMP-4 was found in patients with successful AVF (P = .06). CONCLUSIONS: MMP-2 and TIMP-2 levels were different among patients whose AVF matured vs those who did not. Further follow-up studies to determine the predictability of AVF maturation using relative patient serum levels of MMP-2 and TIMP-2 should be performed.

Vein tissue expression of matrix metalloproteinase as biomarker for hemodialysis arteriovenous fistula maturation.

Failure of arteriovenous fistula (AVF) maturation is attributed to impaired vein remodeling. The purpose of this study is to identify whether vein matrix metalloproteinase (MMP) expression and activity is associated with AVF maturation. Patients with renal insufficiency undergoing surgery had their vein segments harvested and snap-frozen at time of AVF construction. Expression of MMP-2, MMP-9, membrane type-1 MMP (MT1-MMP), tissue inhibitor of metallopreoteinases type 2 (TIMP-2), and TIMP-4 were measured using zymography and Western blotting techniques. Of 14 patients enrolled, 9 had successful maturation and 5 had failure of AVF maturation. Significantly higher levels of MT1-MMP (an MMP-2 activator; P = .01), TIMP-2 (an MMP-2 inhibitor; P = .03), MMP-2 latent (P = .02), and MMP-2 total (P = .03) were associated with AVF maturation. There was a trend toward higher levels of TIMP-4 in the successful group (P = .18). These data demonstrate a positive relationship between MMP-2 expression in veins and AVF maturation. MMP-2 could serve as a potential preoperative marker to predict maturation.

Tissue inhibitor of metalloproteinase-2 regulates matrix metalloproteinase-2-mediated endothelial barrier dysfunction and breast cancer cell transmigration through lung microvascular endothelial cells.

Matrix metalloproteinases (MMP) have been implicated in multiple stages of cancer metastasis. Tissue inhibitor of metalloproteinase-2 (TIMP-2) plays an important role in regulating MMP-2 activity. By forming a ternary complex with pro-MMP-2 and its activator MMP-14 on the cell surface, TIMP-2 can either initiate or restrain the cleavage and subsequent activation of MMP-2. Our recent work has shown that breast cancer cell adhesion to vascular endothelial cells activates endothelial MMP-2, promoting tumor cell transendothelial migration (TEM(E)). However, the mechanism of MMP-2 regulation during TEM(E) remains unclear. In the current study, we present evidence that MMP-14 is expressed in both invasive breast cancer cells (MDA-MB-231 and MDA-MB-436) and lung microvascular endothelial cells (HBMVEC-L), whereas TIMP-2 is exclusively expressed and released from the cancer cells. The tumor cell-derived TIMP-2 was further identified as a major determinant of endothelial MMP-2 activity during tumor cell transmigration in the presence of MMP-14. This response was associated with endothelial barrier dysfunction because coculture of MDA-MB-231 or MDA-MB-436 with HBMVEC-L caused a significant decrease in transendothelial electrical resistance concomitantly with endothelial cell-cell junction disruption and tumor cell transmigration. Knockdown of TIMP-2 or inhibition of TIMP-2/MMP-14 attenuated MMP-2-dependent transendothelial electrical resistance response and TEM(E). These findings suggest a novel interactive role of breast cancer cells and vascular endothelial cells in regulating the TIMP-2/MMP-14/MMP-2 pathway during tumor metastasis.

Psoriasin expression in mammary epithelial cells in vitro and in vivo.

We determined, by serial analysis of gene expression (SAGE) analysis of normal and DCIS (ductal carcinoma in situ) mammary epithelial cells, that psoriasin and several other genes implicated in psoriasis are aberrantly expressed in high-grade, comedo DCIS. Real-time PCR, mRNA in situ hybridization, and immunohistochemical analysis of breast carcinomas confirmed that psoriasin is frequently overexpressed in estrogen receptor-negative tumors. To gain insight into regulatory pathways that control psoriasin expression, we developed polyclonal and monoclonal antibodies and investigated mechanisms that may account for elevated levels of psoriasin in DCIS. Here, we report that loss of attachment to extracellular matrix, growth factor deprivation, and confluent conditions dramatically up-regulate psoriasin expression in MCF10A mammary epithelial cells. All of these conditions are characteristic of high-grade DCIS and psoriatic skin lesions; therefore, the same mechanisms may be responsible for increased expression of psoriasin in vitro and in vivo.