Hypercholesterolemia induces angiogenesis and accelerates growth of breast tumors in vivo.

Obesity and metabolic syndrome are linked to an increased prevalence of breast cancer among postmenopausal women. A common feature of obesity, metabolic syndrome, and a Western diet rich in saturated fat is a high level of circulating cholesterol. Epidemiological reports investigating the relationship between high circulating cholesterol levels, cholesterol-lowering drugs, and breast cancer are conflicting. Here, we modeled this complex condition in a well-controlled, preclinical animal model using innovative isocaloric diets. Female severe combined immunodeficient mice were fed a low-fat/no-cholesterol diet and then randomized to four isocaloric diet groups: low-fat/no-cholesterol diet, with or without ezetimibe (cholesterol-lowering drug), and high-fat/high-cholesterol diet, with or without ezetimibe. Mice were implanted orthotopically with MDA-MB-231 cells. Breast tumors from animals fed the high-fat/high-cholesterol diet exhibited the fastest progression. Significant differences in serum cholesterol level between groups were achieved and maintained throughout the study; however, no differences were observed in intratumoral cholesterol levels. To determine the mechanism of cholesterol-induced tumor progression, we analyzed tumor proliferation, apoptosis, and angiogenesis and found a significantly greater percentage of proliferating cells from mice fed the high-fat/high-cholesterol diet. Tumors from hypercholesterolemic animals displayed significantly less apoptosis compared with the other groups. Tumors from high-fat/high-cholesterol mice had significantly higher microvessel density compared with tumors from the other groups. These results demonstrate that hypercholesterolemia induces angiogenesis and accelerates breast tumor growth in vivo.

Urine vascular biomarkers in Sturge-Weber syndrome.

Sturge-Weber syndrome (SWS) consists of a capillary-venous vascular malformation of the brain, skin and eye. Urine vascular biomarkers have been demonstrated to be abnormal in other vascular anomalies and to correlate with clinical severity and progression. The current study investigated the use of urinary matrix metalloproteinase (MMP)-2, MMP-9, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) levels to non-invasively monitor the progression of SWS. Fifty-four urine samples were collected from patients seen at the Hunter Nelson Sturge-Weber Center at Kennedy Krieger Institute. Urine was analyzed for MMP-2, MMP-9, VEGF and bFGF levels and correlated with clinical outcome at the time of urine collection (n = 48) and 1 year following urine collection (n = 22). Analysis revealed that MMP-2 (p = 0.033) and MMP-9 (p = 0.010) were significantly more likely to be present in the urine of SWS subjects compared to controls and that bFGF was significantly more likely to be present at abnormal levels (p = 0.005). MMP-2 correlated with a more severe clinical score at the time of urine collection, while both MMP-2 and MMP-9 levels correlated with greater disease severity at time of collection. bFGF levels correlated with improved clinical score 1 year after urine collection. These results suggest that MMP-2 and MMP-9 levels may be useful in assessing SWS progression, as well as indicating which patients might benefit from more aggressive treatment, while bFGF levels may be useful in judging the efficacy of neurologic treatment in SWS.

Cooperative benefit for the combination of rapamycin and imatinib in tuberous sclerosis complex neoplasia.

Tuberous sclerosis (TS) is a common autosomal-dominant disorder characterized by tumors of the skin, lung, brain, and kidneys. Monotherapy with rapamycin however resulted in partial regression of tumors, implying the involvement of additional pathways. We have previously implicated platelet-derived growth factor-BB in TS-related tumorigenesis, thus providing a rationale for a combination of mTOR/PDGF blockade using rapamycin and imatinib. Here, we test this combination using a well-established preclinical model of cutaneous tumorigenesis in TS, tsc2ang1 cells derived from a skin tumor from a mouse heterozygous for tsc2. Treatment of tsc2ang1 cells with a combination of rapamycin and imatinib led to an inhibition of proliferation compared with either vehicle treatment or treatment with rapamycin or imatinib monotherapy. Combination therapy also led to a decrease in Akt activation. Potent in vivo activity in animal experiments by combination therapy was noted, without toxicity to the animals. Our findings provide a rationale for the combined use of rapamycin and imatinib, both FDA approved drugs, for the treatment of TS.

Urinary MMP-2 and MMP-9 predict the presence of ovarian cancer in women with normal CA125 levels.

OBJECTIVE: To determine whether urinary matrix metalloproteinases (MMPs) predict the presence of ovarian cancer in patients with CA125 levels below the normal threshold of 35U/mL, a critical group of patients for whom no ovarian cancer biomarker is currently available. To determine whether these noninvasive biomarkers provide clinically useful information in the general ovarian cancer patient population as well. METHODS: ELISA analyses and substrate gel electrophoresis detected the levels and activity of urinary MMP-2, MMP-9, MMP-9/neutrophil gelatinase-associated lipocalin (NGAL) complex, and MMP-9 dimer in all ovarian cancer patients (n=97), those with CA125 <35U/mL (n=26) and controls (n=81). RESULTS: In patients with CA125 <35U/mL, receiver-operating characteristic (ROC) area under curve (AUC) analysis demonstrated that either urinary MMP-2 or MMP-9 or NGAL significantly discriminated between controls and ovarian cancer patients with normal CA125. Multivariate logistic regression revealed that the combination of urinary MMP-2 and MMP-9 provided the best diagnostic accuracy when multiplexed. When further multiplexed with age, the diagnostic accuracy of these biomarkers increased to a significant AUC of 0.820. These findings were consistent among the general ovarian cancer population studied as well, where the combination of urinary MMP-2 and MMP-9 multiplexed with age resulted in a highly significant AUC of 0.881. Pearson chi-square analysis revealed that higher urinary levels of either MMP-2 or MMP-9 were strongly associated with the increasing percentage of women with ovarian cancer independent of CA125 levels. CONCLUSION: This study demonstrates the potential utility of urinary MMP-2 and MMP-9 to differentiate between ovarian cancer patients with normal CA125 levels and controls and suggests that urinary MMP-2 and MMP-9 may be a clinically useful aid in the diagnosis of advanced or recurrent ovarian cancer.

Age-dependent loss of MMP-3 in Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare, progressive segmental premature aging disease that includes scleroderma-like skin, progressive joint contracture, and atherosclerosis. Affected individuals die prematurely of heart attacks or strokes. Extracellular matrix dysregulation is implicated as a factor in disease progression. We analyzed messenger RNA and protein levels for matrix metalloproteinases (MMPs)-2,-3, and -9 in HGPS primary human dermal fibroblasts using real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and gelatin zymography. MMP-3 messenger RNA and protein levels decreased significantly with increasing donor age in HGPS fibroblasts but not in controls. MMP-2 messenger RNA also showed a donor age-dependent decrease in HGPS fibroblasts, but levels of secreted protein were unchanged. MMP-9 was similar in HGPS and control cultures. The decreased MMP-3 may represent a shift in the inherent extracellular matrix-degrading proteolytic balance in favor of matrix deposition in HGPS. This metalloproteinase has the potential to serve as a biomarker of therapeutic efficacy when assessing treatments for HGPS.

Correlation between prenatal urinary matrix metalloproteinase activity and the degree of kidney damage in a large animal model of congenital obstructive uropathy.

BACKGROUND/PURPOSE: We aimed to determine whether the profile of matrix metalloproteinase (MMP) activity in fetal urine correlates with the degree of kidney damage in the setting of congenital obstructive uropathy. METHODS: Fetal lambs underwent either a sham operation or creation of a complete urinary tract obstruction. Necropsies were performed before term, when urinary MMP profiling was performed by zymography; and kidney damage was assessed histologically by multiple semiquantitative analyses and histomorphometric measurements. RESULTS: There was a significant correlation between inner medullary thickness and MMP-9 (P = .005) and 63-kd MMP-2 (P = .019) activities. In like manner, the only MMPs associated with kidney fibrosis were MMP-9 and 63-kd MMP-2. Matrix metalloproteinase-9 activity was a highly significant independent predictor of the total combined kidney fibrosis score (P < .001) as well as of higher fibrosis grades in each of 6 kidney areas analyzed (all with P < .01). The activity of 63-kd MMP-2 correlated significantly with higher fibrosis in select areas. CONCLUSIONS: In a fetal ovine model, urinary MMP activity correlates with the degree of kidney damage. The presence of MMP-9 (in particular) and that of 63-kd MMP-2 are independent predictors of severity. Prenatal urinary MMP profiling may enhance patient stratification and counseling in the setting of congenital obstructive uropathy.

Prenatal urinary matrix metalloproteinase profiling as a potential diagnostic tool in fetal obstructive uropathy.

BACKGROUND/PURPOSE: The diagnostic evaluation, patient stratification, and prenatal counseling for congenital obstructive uropathy remain sub-optimal. Matrix metalloproteinase (MMP) expression profiles are emerging as a valuable diagnostic tool in assorted disease processes. We sought to determine whether congenital obstructive uropathy impacts MMP expression in fetal urine. METHODS: Fetal lambs (n = 25) were divided in two groups: group I (n = 12) underwent a sham operation and group II (n = 13) underwent creation of a complete urinary tract obstruction. Gelatin zymography panels for 4 MMP species were performed on fetal urine in both groups at comparable times post-operatively. Statistical analysis was by the Fisher's exact test (P < .05). RESULTS: Overall fetal survival was 80% (20/25). A variety of significant differences in MMP expression between the two groups were identified. The following profiles were present only in obstructed animals: any MMP other than MMP-2 (P = .029), including any MMP other than 63 kDa and 65 kDa (P = .009); 2 or more MMPs excluding MMP-2s (0.029); and 3 or more MMPs (P = .029). CONCLUSIONS: Limited matrix metalloproteinase expression is present in the urine of normal ovine fetuses. Fetal obstructive uropathy impacts urinary MMP expression in various distinguishable patterns. Prenatal urinary MMP profiling may become a practical and valuable diagnostic tool in the evaluation of congenital obstructive uropathy.