Exploratory Biomarker Analysis Using Plasma Angiogenesis-Related Factors and Cell-Free DNA in the TRUSTY Study: A Randomized, Phase II/III Study of Trifluridine/Tipiracil Plus Bevacizumab as Second-Line Treatment for Metastatic Colorectal Cancer.

BACKGROUND: The TRUSTY study evaluated the efficacy of second-line trifluridine/tipiracil (FTD/TPI) plus bevacizumab in metastatic colorectal cancer (mCRC). OBJECTIVE: This exploratory biomarker analysis of TRUSTY investigated the relationship between baseline plasma concentrations of angiogenesis-related factors and cell-free DNA (cfDNA), and the efficacy of FTD/TPI plus bevacizumab in patients with mCRC. PATIENTS AND METHODS: The disease control rate (DCR) and progression-free survival (PFS) were compared between baseline plasma samples of patients with high and low plasma concentrations (based on the median value) of angiogenesis-related factors. Correlations between cfDNA concentrations and PFS were assessed. RESULTS: Baseline characteristics (n = 65) were as follows: male/female, 35/30; median age, 64 (range 25-84) years; and RAS status wild-type/mutant, 29/36. Patients in the hepatocyte growth factor (HGF)-low and interleukin (IL)-8-low groups had a significantly higher DCR (risk ratio [95% confidence intervals {CIs}]) than patients in the HGF-high (1.83 [1.12-2.98]) and IL-8-high (1.70 [1.02-2.82]) groups. PFS (hazard ratio {HR} [95% CI]) was significantly longer in patients in the HGF-low (0.33 [0.14-0.79]), IL-8-low (0.31 [0.14-0.70]), IL-6-low (0.19 [0.07-0.50]), osteopontin-low (0.39 [0.17-0.88]), thrombospondin-2-low (0.42 [0.18-0.98]), and tissue inhibitor of metalloproteinase-1-low (0.26 [0.10-0.67]) groups versus those having corresponding high plasma concentrations of these angiogenesis-related factors. No correlation was observed between cfDNA concentration and PFS. CONCLUSION: Low baseline plasma concentrations of HGF and IL-8 may predict better DCR and PFS in patients with mCRC receiving FTD/TPI plus bevacizumab, however further studies are warranted. CLINICAL TRIAL REGISTRATION NUMBER: jRCTs031180122.

Kechuanning Gel Plaster Exerts Anti-inflammatory and Immunomodulatory Effects on Ovalbumin-induced Asthma Model Rats via ERK Pathway.

BACKGROUND: We aimed to evaluate the therapeutic effects of Kechuanning gel plaster on ovalbumin (OVA)-induced rat model of asthma. METHODS: Rats were injected with OVA to induce asthma, and Kechuanning gel plaster was administered after the OVA challenge. The immune cell counts in the bronchial alveolar lavage fluid (BALF) were calculated after Kechuanning gel plaster administration. The levels of immune factors in BALF and serum OVA-specific IgE levels were analyzed. Western blot analysis and immunohistochemistry were carried out to analyze the following proteins: C-FOS, C-JUN, RAS p21 protein activator 1 (RASA1), matrix metalloproteinase 9 (MMP9), RAF1, p-MEK1, tissue inhibitor of metalloproteinase-1 (TIMP1), and p-extracellular signal-regulated kinase 1 (ERK1). RESULTS: Administration of Kechuanning gel plaster led to decreased immune cell counts, inflammatory cytokines (interleukin (IL)-1ß, IL13, and IL17), and OVA-specific IgE expression. Compared to the normal group, the C-FOS, C-JUN, RASA1, MMP9, RAF1, MEK1, TIMP1, and p- ERK1 expressions in the model group were significantly increased, whereas Kechuanning gel plaster administration decreased C-JUN, MMP9, TIMP1, RAF1, MEK1, p-ERK1, C-FOS, and RASA1 protein levels. CONCLUSION: Kechuanning gel plaster exerted its therapeutic effects on OVA-induced asthma model rats through the ERK signaling pathway. Kechuanning gel plaster could be considered as a potential alternative therapeutic agent for the management of asthma.

Changes and clinical significance of serum MMP-9, TIMP-1, COX-2, and immune levels in patients with asthma.

OBJECTIVE: To detect serum metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinases (TIMP-1), cyclooxygenase-2 (COX-2), and T helper cells 1-T helper cells 2 (Th1-Th2) levels in asthma patients and assess their clinical significance. METHODS: A total of 72 patients experiencing acute asthma (acute group), 66 stable asthma patients (stable group), and 60 healthy volunteers (control group) were included in this study. The levels of TIMP-1, COX-2, and Th1-Th2 in patients with acute asthma were measured following treatment with budesonide aerosol inhalation. In addition, the levels of MMP-9, TIMP-1, COX-2 and Th1-Th2 were compared in patients with different severity of acute asthma before and after treatment. RESULTS: The serum levels of MMP-9, TIMP-1, and COX-2 showed an increasing trend in the control, stable, and acute groups, while levels of Th1-Th2 showed a sequential decreasing trend, and the differences were statistically significant. Comparison of lung function indexes among the three groups of patients established a negative correlation between serum MMP-9 and its forced vital capacity% predicted (FEV%pred), TIMP-1, and COX-2, and FEV%pred and forced expiratory volume in 1 s-forced vital capacity (FEV1/FVC) levels, but a positive correlation between Th1-Th2 and FEV1/FVC levels in the acute group. A significant difference was observed on comparing the levels of serum MMP-9, TIMP-1, COX-2, and Th1-Th2 in patients with different conditions in the acute group. Specifically, as the condition worsened, a significant increase in serum MMP-9, TIMP-1, and COX-2 levels but a significant decrease in Th1-Th2 levels was observed. After treatment, we observed a significant decrease in serum MMP-9, TIMP-1, and COX-2 levels but a significant increase in Th1-Th2 levels in the acute group. CONCLUSION: The serum levels of MMP-9, TIMP-1, COX-2, and Th1-Th2 are valuable indicators reflecting the condition of asthma patients and could be considered promising clinical monitoring indicators.

Clinical Efficacy of Bushen Yiqi Fuzheng Decoction Combined with Sunitinib in the Treatment of Postoperative Patients with Renal Cell Carcinoma and Its Influence on Their Immune Function.

BACKGROUND: This study aimed to investigate the effect of Bushen Yiqi Fuzheng decoction combined with sunitinib on the prognosis, clinical efficacy and immune function of patients with renal cell carcinoma (RCC) after surgery. METHODS: A total of 120 patients who experienced RCC after surgery were randomly divided into the observation and control groups in this prospective study, with 60 cases in each group. The therapeutic effect, improvement of clinical symptoms, changes of immune function-related indicators and adverse reactions during medication were recorded. The changes in immune cell population, midkine (MK), interleukin 35 (IL-35), hypoxia-inducible factor 2alpha (HIF-2α), matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), carcinoembryonic antigen (CEA), osteopontin (OPN), ferritin (FERR) and beta2-microglobulin (ß2-MG) levels were measured. The Karnofsky performance status (KPS) score of patients was recorded. RESULTS: The total effective rate of the observation group (95%) was better than that of the control group (85%, p < 0.05). After treatment, the changes of immune function indexes in the control group were not obvious. The indexes related to immune function in the observation group significantly decreased. Significant differences were observed in the cluster of differentiation 3+ (CD3+), cluster of differentiation 4+ (CD4+), cluster of differentiation 8+ (CD8+) and CD4+/CD8+ between the two groups after treatment. The incidence of adverse reactions in the observation group was lower than that of the control group. The KPS of the observation group was higher than that of the control group. Before treatment, no differences were observed in the MK, IL-35, HIF-2α, CEA, OPN, FERR, ß2-MG, MMP-9 and TIMP-1 levels between the two groups. After treatment, the levels of the above parameters were lower than those before treatment, especially in the observation group. CONCLUSIONS: Bushen Yiqi Fuzheng decoction combined with sunitinib can significantly improve the clinical efficacy and postoperative immune function of RCC patients after surgery and down-regulate MMP-9 and TIMP-1 levels in the serum, which is beneficial to the prognosis of patients.

Regorafenib prevents the development of emphysema in a murine elastase model.

Emphysema is a chronic obstructive lung disease characterized by inflammation and enlargement of the air spaces. Regorafenib, a potential senomorphic drug, exhibited a therapeutic effect in porcine pancreatic elastase (PPE)-induced emphysema in mice. In the current study we examined the preventive role of regorafenib in development of emphysema. Lung function tests and morphometry showed that oral administration of regorafenib (5 mg/kg/day) for seven days after instillation of PPE resulted in attenuation of emphysema. Mechanistically, regorafenib reduced the recruitment of inflammatory cells, particularly macrophages and neutrophils, in bronchoalveolar lavage fluid. In agreement with these findings, measurements using a cytokine array and ELISA showed that expression of inflammatory mediators including interleukin (IL)-1ß, IL-6, and CXCL1/KC, and tissue inhibitor of matrix metalloprotease-1 (TIMP-1), was downregulated. The results of immunohistochemical analysis confirmed that expression of IL-6, CXCL1/KC, and TIMP-1 was reduced in the lung parenchyma. Collectively, the results support the preventive role of regorafenib in development of emphysema in mice and provide mechanistic insights into prevention strategies. [BMB Reports 2023; 56(8): 439-444].

The effect of early radiofrequency turbinate reduction, intranasal steroid, and antihistamine H-1 on persistent allergic rhinitis: a randomized clinical trial

Abstract Objective: We aimed to evaluate the effect of radiofrequency turbinate reduction as an initial treatment on clinical improvement, inflammatory mediators, and remodeling process. Methods: Between July 2018- February 2020, 32 patients with moderate-severe persistent AR were randomly divided into 2 groups. Intervention group received radiofrequency turbinate reduction followed by intranasal steroid and Antihistamine H-1 (AH-1), control group received intranasal steroid and AH-1. Both groups were evaluated for clinical improvement (using visual analogue scale based on total nasal symptoms score, peak nasal inspiratory flow, and turbinate size using imageJ) after 4 and 8 weeks of treatment. Inflammatory mediators (ELISA from nasal secretions was performed to measure ECP, IL-5, and HSP-70) and remodeling markers (nasal biopsy followed by immunohistochemistry examination was performed to evaluate MMP-9, TIMP-1, and PAI-1) were evaluated in week 4. Results: Three patients dropped out of the study, resulting in 16 patients in intervention group and 13 patients in control group. At week 4, clinical response improved significantly in the intervention group compared to control group (Chi-Square test, p<0.05). Compared to control, intervention group experienced a reduction of IL-5 and no significant change in ECP level (Mann Whitney test, p>0.05). Reduction in the ratio of MMP-9/TIMP-1 were significantly higher in intervention group (unpaired t-test, p< 0,05). Meanwhile, increase in HSP-70 in the intervention group was slightly lower than in control group, but the difference with control group was not significant (Mann Whitney test, p>0.05). Conclusion: Early radiofrequency turbinate reduction followed by pharmacotherapy given to persistent moderate-severe AR patients give more improvement only in early clinical symptoms and reduce MMP-9/TIMP-1 ratio, thus it might be suggested as one of the adjuvant therapies for the management of moderate-severe persistent AR. However, further investigation with a larger sample size and longer follow-up period is needed. Level of evidence: 1B.

Biomarker Development Trial of Satraplatin in Patients with Metastatic Castration-Resistant Prostate Cancer.

BACKGROUND: In the phase III SPARC trial, satraplatin, an oral platinum analogue, demonstrated anticancer activity in men with metastatic castration-resistant prostate cancer (mCRPC). Repeat biopsies are uncommon in mCRPC, limiting the feasibility of tissue-based biomarkers. This phase II study sought to evaluate the feasibility and utility of blood-based biomarkers to identify platinum-sensitive mCRPC. METHODS: Patients with mCRPC who had progressed on docetaxel were enrolled at a single center from 2011 to 2013. Subjects received satraplatin 80 mg/m2 by mouth daily on days 1-5 and prednisone 5 mg PO twice daily, on a 35-day cycle. Serial peripheral blood samples were collected for biomarker assessment. RESULTS: Thirteen docetaxel-refractory mCRPC patients were enrolled, with a median age of 69 years (range 54-77 years) and median PSA of 71.7 ng/mL (range 0.04-3057). Four of 13 patients (31%) responded to satraplatin (defined as a PSA decline of ≥30%). Responders demonstrated improved time to disease progression (206 vs. 35 days, HR 0.26, 95% CI, 0.02-0.24, P = .003). A 6-gene peripheral blood RNA signature and serum tissue inhibitor of metalloproteinase-1 (TIMP-1) levels were assessed as biomarkers, but neither was significantly associated with response to satraplatin. CONCLUSION: In this small series, one-third of mCRPC patients responded to platinum-based chemotherapy. Peripheral blood biomarker measurement is feasible in mCRPC, though the biomarkers we investigated were not associated with platinum response. Other biomarkers, such as DNA damage repair mutations, should be evaluated.

Intra-articular injection of rAAV-hFGF-2 ameliorates monosodium iodoacetate-induced osteoarthritis in rats via inhibiting TLR-4 signaling and activating TIMP-1.

Osteoarthritis (OA) is a chronic debilitating degenerative disorder leading to structural, and functional anomaly of the joint. The present study tests the hypothesis that overexpression of the basic fibroblast growth factor (FGF-2) via direct rAAV-mediated gene transfer suppresses monosodium iodoacetate (MIA)-induced knee OA in rats relative to control (reporter rAAV-lacZ vector) gene transfer by intra-articular injection. Rats were treated with 20 µl rAAV-hFGF-2 on weekly basis; on days 7, 14, and 21 after single intra-articular injection of MIA (3 mg/50 µl saline). FGF-2 reduced knee joint swelling and improved motor performance and muscle coordination as evidenced by increased distance travelled, mean speed, rearing frequency in open field test (OFT) as well as fall-off latency in rotarod test together with reduced immobility time in OFT. Moreover, FGF-2 attenuated MIA-related radiological and histological alterations. Indeed, FGF-2 decreased knee joint inflammatory biomarker as demonstrated by reduced mRNA expression of toll like receptor (TLR)-4 and its downstream mediators such as tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1ß) and high motility group box (HMGB) 1. In parallel, FGF-2 attenuated knee joint degradation biomarkers as reflected by the downregulation of ADAMTS-5 mRNA expression and matrix metalloproteinase 13 (MMP-13) content together with the up-regulation of tissue inhibitor of metalloproteinase (TIMP)-1 mRNA expression. These findings suggest a potential therapeutic role for FGF-2 against MIA-induced knee OA in rats via inhibition of TLR4 signaling and activating TIMP-1, resulting in down-regulation of ADAMTS-5 and MMP-13.

The effect of early radiofrequency turbinate reduction, intranasal steroid, and antihistamine H-1 on persistent allergic rhinitis: a randomized clinical trial.

OBJECTIVE: We aimed to evaluate the effect of radiofrequency turbinate reduction as an initial treatment on clinical improvement, inflammatory mediators, and remodeling process. METHODS: Between July 2018-February 2020, 32 patients with moderate-severe persistent AR were randomly divided into 2 groups. Intervention group received radiofrequency turbinate reduction followed by intranasal steroid and Antihistamine H-1 (AH-1), control group received intranasal steroid and AH-1. Both groups were evaluated for clinical improvement (using visual analogue scale based on total nasal symptoms score, peak nasal inspiratory flow, and turbinate size using imageJ) after 4 and 8 weeks of treatment. Inflammatory mediators (ELISA from nasal secretions was performed to measure ECP, IL-5, and HSP-70) and remodeling markers (nasal biopsy followed by immunohistochemistry examination was performed to evaluate MMP-9, TIMP-1, and PAI-1) were evaluated in week 4. RESULTS: Three patients dropped out of the study, resulting in 16 patients in intervention group and 13 patients in control group. At week 4, clinical response improved significantly in the intervention group compared to control group (Chi-Square test, p < 0.05). Compared to control, intervention group experienced a reduction of IL-5 and no significant change in ECP level (Mann Whitney test, p > 0.05). Reduction in the ratio of MMP-9/TIMP-1 were significantly higher in intervention group (unpaired t-test, p < 0,05). Meanwhile, increase in HSP-70 in the intervention group was slightly lower than in control group, but the difference with control group was not significant (Mann Whitney test, p > 0.05). CONCLUSION: Early radiofrequency turbinate reduction followed by pharmacotherapy given to persistent moderate-severe AR patients give more improvement only in early clinical symptoms and reduce MMP-9/TIMP-1 ratio, thus it might be suggested as one of the adjuvant therapies for the management of moderate-severe persistent AR. However, further investigation with a larger sample size and longer follow-up period is needed. LEVEL OF EVIDENCE: 1B.

[Lutein inhibits the adhesion, invasiveness and metastasis of human prostate cancer PC-3M cells].

OBJECTIVE: To explore the effects of lutein on the adhesion, invasiveness and metastasis of human prostate cancer PC-3M cells and its action mechanism. METHODS: We divided human prostate cancer PC-3M cells into a control, a low-dose lutein, a medium-dose lutein and a high-dose lutein group, and treated them with 0, 10, 20 and 40 µmol/L lutein, respectively. Then we examined the adhesion of the cells to matrix by cell adhesion assay and the changes in cell pseudopodia by Phalloidin staining, detected the expressions of paxillin, matrix metalloproteinase 2 (MMP-2), MMP-9, recombinant tissue inhibitors of metalloproteinase 1 (TIMP-1), E-cadherin, N-cadherin and vimentin by Western blot, determined the invasiveness and migration of the cells by scratch and Transwell assays, and observed their dynamic movement by high-intension imaging. RESULTS: Compared with the control, the lutein intervention groups showed significant reduction in the number of the cells adhered to matrix, the number of cell pseudopodia, the expressions of paxillin, MMP-2, MMP-9, N-cadherin and vimentin, the rates of migration, invasion and metastasis, and the distances of displacement and movement of the cells. However, the expressions of TIMP-1 and epithelial-mesenchymal transition-related E-cadherin were upregulated significantly. CONCLUSION: Lutein can inhibit cell adhesion, reduce the expressions of MMPs, and suppress cell invasion and migration by inhibiting the process of epithelial-mesenchymal transition.

24-month decline of non-invasive liver fibrosis markers in HCV-mono and HCV/HIV coinfection after direct-acting antiviral therapy.

Long term liver fibrosis (LF) changes and their best -monitoring non-invasive markers (NILFM) after effective anti-HCV DAA therapy are little- known. Matrix-metalloproteases (MMPs) and their tissue-inhibitors (TIMPs) are pivotal in liver inflammation repair. Their plasma levels might assess long-term LF changes after therapy. Overall 374 HCV-infected adult patients, 214 HCV-HIV coinfected, were followed-up for 24 months after starting DAA. LF was assessed by transient elastometry (TE), biochemical indexes (APRI, Forns, FIB-4) and, in 61 individuals, by MMPs and TIMP-1 plasma levels. Several MMPs and TIMP-1 SNPs were genotyped in 319 patients. TE was better than biochemical indexes for early and long-term LF monitoring. MMPs-2,-8,-9 and-TIMP-1 levels and TE displayed parallel declining curves although only TIMP-1 correlated with TE (P = 0.006) and biochemical indexes (P < 0.02). HCV monoinfected had significantly higher baseline NILFM and TIMP-1 plasma values, but lower MMPs levels than coinfected patients. No differences in NILFM course were observed between mono-and coinfected or between different DAA regimens. Only the MMP-2 (-1306 C/T) variant TT genotype associated with higher values of NILFM NILFM decline extends 24 months after therapy. TE and TIMP1 are reliable LF-monitoring tools. NILFM courses were similar in mono-and coinfected patients, DAA regimens type did not influence NILFM course.

Effect of combined farnesoid X receptor agonist and angiotensin II type 1 receptor blocker on ongoing hepatic fibrosis.

OBJECTIVE: Nonalcoholic steatohepatitis (NASH) is difficult to diagnose in patients with no symptoms. We aimed to investigate the combined effect of farnesoid X receptor (FXR) agonist, obeticholic acid (OCA), and angiotensin II type 1 receptor blocker (ARB: losartan) on an ongoing hepatic fibrosis in a NASH rat model. METHODS: Fischer 344 rats were fed with choline-deficient L-amino-acid-defined (CDAA) diet for 16 weeks. After 8-week administration of CDAA diet, OCA, losartan, or a combination of these drugs was administered at a dose of 30 mg/kg/day for 8 weeks by oral gavage. The in vivo and in vitro effects of OCA + losartan and liver fibrosis progression, lipopolysaccharide (LPS), Toll-like receptor 4 (TLR4) regulatory cascade, and gut barrier function were evaluated. RESULTS: OCA + losartan alleviated hepatic fibrosis progression by suppressing α-SMA expression. It inhibited the proliferation of activated hepatic stellate cell (Ac-HSC) and mRNA expression of hepatic transforming growth factor-ß1 (TGF-ß1), TLR4, and tissue inhibitor of metalloproteinase-1 (TIMP-1) and decreased the hydroxyproline levels. OCA increased the hepatic matrix metalloproteinase-2 (MMP-2) mRNA expression. OCA decreased the mRNA expression of hepatic LPS-binding protein and intestinal permeability by ameliorating the disruption of CDAA diet-induced zonula occludens-1. Losartan directly inhibited the proliferation of Ac-HSC. The in vitro suppressive effects of OCA + losartan on the mRNA expressions of TGF-ß1 and α1(I)-procollagen, TLR4, and TIMP-1 in Ac-HSCs were almost in parallel. CONCLUSIONS: OCA + losartan suppressed the ongoing hepatic fibrosis by attenuating gut barrier dysfunction and suppressing Ac-HSC proliferation. Combined therapy may be a promising novel approach for NASH with fibrosis.

Exploring the angiographic-biologic phenotype in the IMAGINE study: quantitative UWFA and cytokine expression.

BACKGROUND: This study investigates the association of intraocular cytokine expression and ultrawide-field fluorescein angiography (UWFA) quantitative imaging biomarkers and their association with angiographical feature response after antivascular endothelial growth factor (VEGF) therapy in diabetic macular oedema (DME). METHODS: The IMAGINE DME study is a post hoc imaging biomarker and intraocular cytokine assessment from the DAVE study, a prospective DME clinical trial that included aqueous humour sampling and UWFA imaging. Fifty-four cytokines associated with inflammation and angiogenesis were evaluated through multiplex arrays. UWFA parameters were assessed using an automated feature analysis platform to determine ischaemic and leakage indices and microaneurysm (MA) count. Eyes were classified into UWFA responder or non-responder groups based on longitudinal quantitative UWFA parameter improvement. Cytokine expression was correlated with UWFA metrics and evaluated in the context of therapeutic response. RESULTS: Twenty-one eyes were included with a mean age of 55±10 years. Increased panretinal leakage index correlated with VEGF (r=0.70, p=0.0005), angiopoietin-like 4 (r=0.77, p=4.6E-5) and interleukin (IL)-6 (r=0.64, p=0.002). Panretinal ischaemic index was associated with tissue inhibitor of metalloproteinases 1 (TIMP-1, r=0.49, p=0.03) and peripheral ischaemia correlated with VEGF (r=0.45, p=0.05). MA count correlated with increased monocyte chemotactic protein-4 (MCP-4, r=0.60, p=0.004) and platelet and endothelial cell adhesion molecule 1 (PECAM-1, r=0.58, p=0.005). Longitudinal MA reduction was associated with decreased baseline VEGF and urokinase receptor (uPAR) (p<0.05). High baseline VEGF and IL-6 were associated with dramatic reduction in macular leakage (p<0.05). CONCLUSIONS: Baseline and longitudinal quantitative UWFA imaging parameters correlated with multiple aqueous humour cytokine concentrations, including VEGF and IL-6. Further research is needed to assess the possible implications of using these findings for evaluating treatment response.

Circulating TIMP-1 is associated with hematoma volume in patients with spontaneous intracranial hemorrhage.

Matrix metalloproteinases (MMPs) are proteolytic zinc-endopeptidases regulated by tissue Inhibitors of matrix metalloproteinases (TIMPs). We evaluated the potential of MMPs and TIMPs as clinical tools for Intracranial Haemorrhage (ICH). Spontaneous non-traumatic ICH patients were recruited from two hospitals: Complejo Hospitalario de Navarra (CHN = 29) and Vall d´Hebron (VdH = 76). Plasmatic levels of MMP-1, -2, -7, -9, -10 and TIMP-1 and their relationship with clinical, radiological and functional variables were evaluated. We further studied the effect of TIMP-1 (0.05-0.2 mg/Kg) in an experimental tail-bleeding model. In CHN, TIMP-1 was associated with admission-hematoma volume and MMP-7 was elevated in patients with deep when compared to lobar hematoma. In VdH, admission-hematoma volume was associated with TIMP-1 and MMP-7. When data from both hospitals were combined, we observed that an increase in 1 ng/ml in TIMP-1 was associated with an increase of 0.14 ml in haemorrhage (combined ß = 0.14, 95% CI = 0.08-0.21). Likewise, mice receiving TIMP-1 (0.2 mg/Kg) showed a shorter bleeding time (p < 0.01). Therefore, the association of TIMP-1 with hematoma volume in two independent ICH cohorts suggests its potential as ICH biomarker. Moreover, increased TIMP-1 might not be sufficient to counterbalance MMPs upregulation indicating that TIMP-1 administration might be a beneficial strategy for ICH.

Gelatinases and their tissue inhibitors in a group of subjects with obstructive sleep apnea syndrome.

Obstructive sleep apnea syndrome (OSAS) is associated with an elevated risk of cardiovascular events and stroke. Matrix metalloproteinases (MMPs) are endopeptidases involved in extracellular matrix degradation and then in the development and progression of cardiovascular diseases. Our aim was to evaluate plasma levels of gelatinases (MMP-2 and MMP-9) and their tissue inhibitors (TIMP-1 and TIMP-2) in a group of subjects with OSAS. We enrolled 48 subjects (36 men and 12 women; mean age 49.7 ± 14.68 yrs) with OSAS diagnosed with a 1-night cardiorespiratory study and then we subdivided these subjects into two subgroups according to the apnea/hypopnea index (AHI): Low (L = 21 subjects with AHI <30) and High (H = 27 subjects with AHI >30). We measured plasma concentration of the gelatinases and their inhibitors using ELISA kits. We observed a significant increase in plasma concentration of MMP-9, MMP-2, TIMP-1 and TIMP-2 in the entire group of OSAS subjects and in the two subgroups, with higher levels in the H in comparison with the L subgroup. In the whole group of OSAS subjects we also noted a significant decrease in MMP-9/TIMP-1 ratio in comparison with normal controls. Only MMP-9 was significantly correlated with the severity of the disease, expressed as AHI, with the oxygen desaturation index and also with the mean oxygen saturation. MMPs pattern is altered in OSAS and significantly influenced by the severity of the disease; it probably contributes to the vascular remodeling that leads to the atherosclerotic disease and cardiovascular complications.

Matrix metalloproteinases and their tissue inhibitor after reperfused ST-elevation myocardial infarction treated with doxycycline. Insights from the TIPTOP trial.

BACKGROUND: The TIPTOP (Early Short-term Doxycycline Therapy In Patients with Acute Myocardial Infarction and Left Ventricular Dysfunction to Prevent The Ominous Progression to Adverse Remodelling) trial demonstrated that a timely, short-term therapy with doxycycline is able to reduce LV dilation, and both infarct size and severity in patients treated with primary percutaneous intervention (pPCI) for a first ST-elevation myocardial infarction (STEMI) and left ventricular (LV) dysfunction. In this secondary, pre-defined analysis of the TIPTOP trial we evaluated the relationship between doxycycline and plasma levels of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). METHODS: In 106 of the 110 (96%) patients enrolled in the TIPTOP trial, plasma MMPs and TIMPs were measured at baseline, and at post-STEMI days 1, 7, 30 and 180. To evaluate the remodeling process, 2D-Echo studies were performed at baseline and at 6months. A (99m)Tc-SPECT was performed to evaluate the 6-month infarct size and severity. RESULTS: Doxycycline therapy was independently related to higher plasma TIMP-2 levels at day 7 (p<0.05). Plasma TIMP-2 levels above the median value at day 7 were correlated with the 6-month smaller infarct size (3% [0%-16%] vs. 12% [0%-30%], p=0.002) and severity (0.55 [0.44-0.64] vs. 0.45 [0.29-0.60], p=0.002), and LV dilation (-1ml/m(2) [from -7ml/m(2) to 9ml/m(2)] vs. 3ml/m(2) [from -2ml/m(2) to 19ml/m(2)], p=0.04), compared to their counterpart. CONCLUSIONS: In this clinical setting, doxycycline therapy results in higher plasma levels of TIMP-2 which, in turn, inversely correlate with 6month infarct size and severity as well as LV dilation.

Antifibrotic effects of a recombinant adeno-associated virus carrying small interfering RNA targeting TIMP-1 in rat liver fibrosis.

Elevated tissue inhibitor of metalloproteinase 1 (TIMP-1) expression contributes to excess production of extracellular matrix in liver fibrosis. Herein, we constructed a recombinant adeno-associated virus (rAAV) carrying siRNA of the TIMP-1 gene (rAAV/siRNA-TIMP-1) and investigated its effects on liver fibrosis in rats. Two models of rat liver fibrosis, the carbon tetrachloride and bile duct ligation models, were treated with rAAV/siRNA-TIMP-1. In the carbon tetrachloride model, rAAV/siRNA-TIMP-1 administration attenuated fibrosis severity, as determined by histologic analysis of hepatic collagen accumulation, hydroxyproline content, and concentrations of types I and III collagen in livers and sera. Levels of mRNA and active matrix metalloproteinase (MMP) 13 were elevated, whereas levels of mRNA and active MMP-2 were decreased. Moreover, a marked decrease was noted in the expression of α-smooth muscle actin, a biomarker of activated hepatic stellate cells (HSCs), and transforming growth factor-ß1, critical for the development of liver fibrosis. Similarly, rAAV/siRNA-TIMP-1 treatment significantly alleviated bile duct ligation-induced liver fibrosis. Furthermore, this treatment dramatically suppressed TIMP-1 expression in HSCs from both model rats. These data indicate that the administration of rAAV/siRNA-TIMP-1 attenuated liver fibrosis by directly elevating the function of MMP-13 and diminishing activated HSCs. It also resulted in indirect decreased expression of type I collagen, MMP-2, and transforming growth factor-ß1. In conclusion, rAAV/siRNA-TIMP-1 may be an effective antifibrotic gene therapy agent.

Potential role of metalloproteinase inhibitors from radiation­sterilized amnion dressings in the healing of venous leg ulcers.

Chronic wounds are a significant socio-economic problem, thus, the improvement of the effectiveness of their treatment is an important objective for public health strategies. The predominant stage of the chronic wound is the inflammatory reaction which is associated with the damage of tissues, possibly due to the excessive secretion and activation of matrix metalloproteinases (MMPs). Several reports have suggested that amnion dressing inhibits tissue destruction and accelerates wound healing. Our recent study revealed that sterilized amnion stimulates keratinocyte proliferation in vitro, while the present study focused on the clinical application of radiation-sterilized amnion in chronic venous leg ulcers and aimed to explain the possible mechanism of its in vivo action. The study involved 25 individuals suffering from venous leg ulceration with a surface area of 10-100 cm2 and a healing rate below 10% per week, as verified during a 2-week screening period. The effectiveness of the amnion dressing was estimated following 4 weeks of treatment. The wound assessment, based on a modified Bates-Jensen Questionnaire, revealed a good and satisfactory response to the treatment in 23 of the 25 patients. The measurement of MMP-2 and MMP-9 activities in wound exudates revealed a decrease in activity in response to amnion application. This effect resulted from the presence of the potent MMP inhibitors, tissue inhibitor of metalloproteinases-1 (TIMP-1), type-1 plasminogen activator inhibitor (PAI-1) and thrombospondin-1 (TSP-1) in the amnion dressings, as shown by real-time fluorescence zymography and protein microarrays. Thus, unlike modern synthetic dressing materials, radiation-sterilized amnion dressings may have a multidirectional beneficial effect on chronic wounds.

Injection of AAV2-BMP2 and AAV2-TIMP1 into the nucleus pulposus slows the course of intervertebral disc degeneration in an in vivo rabbit model.

BACKGROUND CONTEXT: Intervertebral disc degeneration (IDD) is a common cause of back pain. Patients who fail conservative management may face the morbidity of surgery. Alternative treatment modalities could have a significant impact on disease progression and patients' quality of life. PURPOSE: To determine if the injection of a virus vector carrying a therapeutic gene directly into the nucleus pulposus improves the course of IDD. STUDY DESIGN: Prospective randomized controlled animal study. METHODS: Thirty-four skeletally mature New Zealand white rabbits were used. In the treatment group, L2-L3, L3-L4, and L4-L5 discs were punctured in accordance with a previously validated rabbit annulotomy model for IDD and then subsequently treated with adeno-associated virus serotype 2 (AAV2) vector carrying genes for either bone morphogenetic protein 2 (BMP2) or tissue inhibitor of metalloproteinase 1 (TIMP1). A nonoperative control group, nonpunctured sham surgical group, and punctured control group were also evaluated. Serial magnetic resonance imaging (MRI) studies at 0, 6, and 12 weeks were obtained, and a validated MRI analysis program was used to quantify degeneration. The rabbits were sacrificed at 12 weeks, and L4-L5 discs were analyzed histologically. Viscoelastic properties of the L3-L4 discs were analyzed using uniaxial load-normalized displacement testing. Creep curves were mathematically modeled according to a previously validated two-phase exponential model. Serum samples obtained at 0, 6, and 12 weeks were assayed for biochemical evidence of degeneration. RESULTS: The punctured group demonstrated MRI and histologic evidence of degeneration as expected. The treatment groups demonstrated less MRI and histologic evidence of degeneration than the punctured group. The serum biochemical marker C-telopeptide of collagen type II increased rapidly in the punctured group, but the treated groups returned to control values by 12 weeks. The treatment groups demonstrated several viscoelastic properties that were distinct from control and punctured values. CONCLUSIONS: Treatment of punctured rabbit intervertebral discs with AAV2-BMP2 or AAV2-TIMP1 helps delay degenerative changes, as seen on MRI, histologic sampling, serum biochemical analysis, and biomechanical testing. Although data from animal models should be extrapolated to the human condition with caution, this study supports the potential use of gene therapy for the treatment of IDD.