Validity and Reliability of Value Assessment Frameworks for New Cancer Drugs.

BACKGROUND: Several organizations have developed frameworks to systematically assess the value of new drugs. These organizations include the American Society of Clinical Oncology (ASCO), the European Society for Medical Oncology (ESMO), the Institute for Clinical and Economic Review (ICER), and the National Comprehensive Cancer Network (NCCN). OBJECTIVES: To understand the extent to which these four tools can facilitate value-based treatment decisions in oncology. METHODS: In this pilot study, eight panelists conducted value assessments of five advanced lung cancer drugs using the ASCO, ESMO, and ICER frameworks. The panelists received instructions and published clinical data required to complete the assessments. Published NCCN framework scores were abstracted. The Kendall's W coefficient was used to measure convergent validity among the four frameworks. Intraclass correlation coefficients were used to measure inter-rater reliability among the ASCO, ESMO, and ICER frameworks. Sensitivity analyses were conducted. RESULTS: Drugs were ranked similarly by the four frameworks, with Kendall's W of 0.703 (P = 0.006) across all the four frameworks. Pairwise, Kendall's W was the highest for ESMO-ICER (W = 0.974; P = 0.007) and ASCO-NCCN (W = 0.944; P = 0.022) and the lowest for ICER-NCCN (W = 0.647; P = 0.315) and ESMO-NCCN (W = 0.611; P = 0.360). Intraclass correlation coefficients (confidence interval [CI]) for the ASCO, ESMO, and ICER frameworks were 0.786 (95% CI 0.517-0.970), 0.804 (95% CI 0.545-0.973), and 0.281 (95% CI 0.055-0.799), respectively. When scores were rescaled to 0 to 100, the ICER framework provided the narrowest band of scores. CONCLUSIONS: The ASCO, ESMO, ICER, and NCCN frameworks demonstrated convergent validity, despite differences in conceptual approaches used. The ASCO inter-rater reliability was high, although potentially at the cost of user burden. The ICER inter-rater reliability was poor, possibly because of its failure to distinguish differential value among the sample of drugs tested. Refinements of all frameworks should continue on the basis of further testing and stakeholder feedback.

Follow-up intervals in patients with Cushing's disease: recommendations from a panel of experienced pituitary clinicians.

PURPOSE: Follow-up guidelines are needed to assess quality of care and to ensure best long-term outcomes for patients with Cushing's disease (CD). The purpose of this study was to assess agreement by experts on recommended follow-up intervals for CD patients at different phases in their treatment course. METHODS: The RAND/UCLA modified Delphi process was used to assess expert consensus. Eleven clinicians who regularly manage CD patients rated 79 hypothetical patient scenarios before and after ("second round") an in-person panel discussion to clarify definitions. Scenarios described CD patients at various time points after treatment. For each scenario, panelists recommended follow-up intervals in weeks. Panel consensus was assigned as follows: "agreement" if no more than two responses were outside a 2 week window around the median response; "disagreement" if more than two responses were outside a 2 week window around the median response. Recommendations were developed based on second round results. RESULTS: Panel agreement was 65.9% before and 88.6% after the in-person discussion. The panel recommended follow-up within 8 weeks for patients in remission on glucocorticoid replacement and within 1 year of surgery; within 4 weeks for patients with uncontrolled persistent or recurrent disease; within 8-24 weeks in post-radiotherapy patients controlled on medical therapy; and within 24 weeks in asymptomatic patients with stable plasma ACTH concentrations after bilateral adrenalectomy. CONCLUSIONS: With a high level of consensus using the Delphi process, panelists recommended regular follow-up in most patient scenarios for this chronic condition. These recommendations may be useful for assessment of CD care both in research and clinical practice.

BIOCHEMICAL CONTROL DURING LONG-TERM FOLLOW-UP OF 230 ADULT PATIENTS WITH CUSHING DISEASE: A MULTICENTER RETROSPECTIVE STUDY.

OBJECTIVE: Cushing disease (CD) results from excessive exposure to glucocorticoids caused by an adrenocorticotropic hormone-secreting pituitary tumor. Inadequately treated CD is associated with significant morbidity and elevated mortality. Multicenter data on CD patients treated in routine clinical practice are needed to assess treatment outcomes in this rare disorder. The study purpose was to describe the burden of illness and treatment outcomes for CD patients. METHODS: Eight pituitary centers in four U.S. regions participated in this multicenter retrospective chart review study. Subjects were CD patients diagnosed at ≥18 years of age within the past 20 years. Descriptive statistical analyses were conducted to examine presenting signs, symptoms, comorbidities, and treatment outcomes. RESULTS: Of 230 patients, 79% were female (median age at diagnosis, 39 years; range, 18 to 78 years). Length of follow-up was 0 to 27.5 years (median, 1.9 years). Pituitary adenomas were 0 to 51 mm. The most common presenting comorbidities included hypertension (67.3%), polycystic ovary syndrome (43.5%), and hyperlipidemia (41.5%). Biochemical control was achieved with initial pituitary surgery in 41.4% patients (91 of 220), not achieved in 50.0% of patients (110 of 220), and undetermined in 8.6% of patients (19 of 220). At the end of follow-up, control had been achieved with a variety of treatment methods in 49.1% of patients (110 of 224), not achieved in 29.9% of patients (67 of 224), and undetermined in 21.0% of patients (47 of 224). CONCLUSION: Despite multiple treatments, at the end of follow-up, biochemical control was still not achieved in up to 30% of patients. These multicenter data demonstrate that in routine clinical practice, initial and long-term control is not achieved in a substantial number of patients with CD. ABBREVIATIONS: BLA = bilateral adrenalectomy CD = Cushing disease CS = Cushing syndrome eCRF = electronic case report form MRI = magnetic resonance imaging PCOS = polycystic ovary syndrome.

ShinyFruit: interactive fruit phenotyping software and its application in blackberry.

Introduction: Horticultural plant breeding programs often demand large volumes of phenotypic data to capture visual variation in quality of harvested products. Increasing the throughput potential of phenomic pipelines enables breeders to consider data-hungry molecular breeding strategies such as genome-wide association studies and genomic selection. Methods: We present an R-based web application called ShinyFruit for image-based phenotyping of size, shape, and color-related qualities in fruits and vegetables. Here, we have demonstrated one potential application for ShinyFruit by comparing its estimates of fruit length, width, and red drupelet reversion (RDR) with ImageJ and analogous manual phenotyping techniques in a population of blackberry cultivars and breeding selections from the University of Arkansas System Division of Agriculture Fruit Breeding Program. Results: ShinyFruit results shared a strong positive correlation with manual measurements for blackberry length (r = 0.96) and ImageJ estimates of RDR (r = 0.96) and significant, albeit weaker, correlations with manual RDR estimation methods (r = 0.62 - 0.70). Neither phenotyping method detected genotypic differences in blackberry fruit width, suggesting that this trait is unlikely to be heritable in the population observed. Discussion: It is likely that implementing a treatment to promote RDR expression in future studies might strengthen the documented correlation between phenotyping methods by maximizing genotypic variance. Even so, our analysis has suggested that ShinyFruit provides a viable, open-source solution to efficient phenotyping of size and color in blackberry fruit. The ability for users to adjust analysis settings should also extend its utility to a wide range of fruits and vegetables.

The Association of Choroidal Thickening by Atropine With Treatment Effects for Myopia: Two-Year Clinical Trial of the Low-concentration Atropine for Myopia Progression (LAMP) Study.

PURPOSE: To evaluate longitudinal changes in subfoveal choroidal thickness (SFChT) among children receiving atropine 0.05%, 0.025%, or 0.01% over 2 years and their associations with treatment outcomes in myopia control. DESIGN: Double-blinded randomized controlled trial. METHODS: SFChT was measured at 4-month intervals using spectral domain optical coherence tomography. Cycloplegic spherical equivalent (SE), axial length (AL), best-corrected visual acuity, parental SE, outdoor time, near work diopter hours, and treatment compliance were also measured. RESULTS: 314 children were included with qualified choroidal data. The 2-year changes in SFChT from baseline were 21.15 ± 32.99 µm, 3.34 ± 25.30 µm, and -0.30 ± 27.15 µm for the atropine 0.05%, 0.025%, and 0.01% groups, respectively (P < .001). A concentration-dependent response was observed, with thicker choroids at higher atropine concentrations (ß = 0.89, P < .001). Mean SFChT thickness significantly increased at 4 months in the atropine 0.025% (P = .001) and 0.05% groups (P < .001) and then remained stable until the end of the second year (P > .05 for all groups). Over 2 years, an increase in SFChT was associated with slower SE progression (ß = 0.074, P < .001) and reduced AL elongation (ß = -0.045, P < .001). In the mediation analysis, 18.45% of the effect on SE progression from atropine 0.05% was mediated via its choroidal thickening. CONCLUSIONS: Low concentration atropine induced a choroidal thickening effect along a concentration-dependent response throughout the treatment period. The choroidal thickening was associated with a slower SE progression and AL elongation among all the treatment groups. Choroidal response can be used for assessment of long-term treatment outcomes and as a guide for concentration titrations of atropine.

Elucidating mechanisms of osteogenesis in human adipose-derived stromal cells via microarray analysis.

INTRODUCTION: The osteogenic potential of human adipose-derived stromal cells (hASCs), the ease of cell procurement, and the shortcomings of conventional skeletal reconstruction call for further analysis of the molecular mechanisms governing hASC osteogenic differentiation. We have examined the expression profile of the human transcriptome during osteogenic differentiation of ASCs using microarray. Subsequently, we analyzed those genes related to osteogenesis that have not been previously studied about hASCs. We have preliminarily assessed the role of IGFBP3, TGF-B3, TNC, CTGF, DKK-1, and PDGFRB in hASC osteogenic differentiation. METHODS: We compared the expression profile of undifferentiated hASCs to that of hASCs treated with osteogenic differentiation medium for 1, 3, or 7 days using the Human Exonic Evidence-Based Oligonucleotide chip. Genes significantly overexpress or underexpressed were validated with quantitative reverse transcription-polymerase chain reaction. The osteogenic capability of ASCs was verified by Alizarin Red staining. RESULTS: IGFBP3, TGF-B3, TNC, CTGF, and PDGFRB were all upregulated in early osteogenesis, and TGF-B3, TNC, and PDGFRB were upregulated in late osteogenesis by microarray and quantitative reverse transcription analysis. In contrast, DKK-1 was downregulated in early and late osteogenesis. Alizarin Red staining showed a significant increase in mineralization in hASCs, even after 1 day in osteogenic differentiation medium. CONCLUSIONS: Factors that commit hASCs to an osteogenic pathway remain largely unknown. We have described 6 genes that play key roles in hASC osteogenic differentiation. We plan to further exploit these data via in vitro treatment of hASCs with these soluble cytokines and in vivo translation using a nude mouse calvarial defect model.

Maternal influences on fruit and vegetable consumption of schoolchildren: case study in Hong Kong.

This study investigated whether the consumption of fruit and vegetable by Chinese primary students in Hong Kong is associated with their mother's (1) nutrition knowledge on fruit and vegetables; (2) attitude towards healthy eating; and (3) fruit and vegetable consumption. Fourth- and fifth-grade students from 10 primary schools located in different districts, along with their mothers, were invited to each fill-in a questionnaire related to their diet intake. Accomplished questionnaires were matched and analysed. There were 1779 mother-child pairs who were successfully matched. Chi-square analysis revealed that students' fruit consumption is associated with their mother's (1) knowledge on fruit and vegetables (P = 0.006); (2) attitude towards healthy eating (P = 0.010); and (3) fruit consumption (P < 0.001). Students' vegetable consumption exhibited the same association with their mother's (1) knowledge (P < 0.001), (2) attitude towards healthy eating (P = 0.005), and (3) vegetable consumption (P < 0.001). Logistic regression showed that knowledge, attitude and dietary practice of mothers were independent factors associated with the consumption of fruit and vegetables by students and are not influenced by the level of education and household income. The results highlight the important role of parents in promoting fruit and vegetable consumption to primary students. It reaffirmed the importance of parent nutrition education in the formulation of a comprehensive health promotion strategy to school-aged children.

Improving nutritional status of children with Cerebral palsy: a qualitative study of caregiver experiences and community-based training in Ghana.

BACKGROUND: Cerebral palsy (CP) is the most common childhood disability worldwide, and evidence shows that children with CP are at an increased risk of malnutrition due to feeding difficulties. This qualitative study explores caregiver experiences of feeding before and after a community-based training program in Ghana. METHODS: Thirteen caregivers of children with CP, who were severely undernourished, were interviewed at the start of the training program. Eleven of these were interviewed again after a year of monthly group trainings and home visits, which included guidance on feeding. Four additional caregivers were interviewed at end line. Interviews explored caregivers' mealtime experiences, as well as a 24-hr dietary recall and a structured feeding observation checklist. Children's nutritional status was assessed by anthropometry. RESULTS: Caregivers found mealtimes stressful due to time demands, messiness, and the pressure of providing enough quality food. They felt that the training program had helped reduced this stress and dietary recall data suggested some improved dietary quality. However, there was neither improvement nor deterioration in anthropometric status of the children. CONCLUSION: Group trainings were welcomed by caregivers and notably reduced stress around feeding times. However, future work is needed in order to improve anthropometric outcomes, including, but not limited to, greater focus on nutritional requirements during caregiver training interventions. Therapeutic feeding programs must also be better utilized and need to be better equipped to care for this group of children, including deviating from standard admission and treatment protocols.

Mouse R-spondin2 is required for apical ectodermal ridge maintenance in the hindlimb.

The R-spondin (Rspo) family of proteins consists of secreted cysteine-rich proteins that can activate beta-catenin signaling via the Frizzled/LRP5/6 receptor complex. Here, we report that targeted inactivation of the mouse Rspo2 gene causes developmental limb defects, especially in the hindlimb. Although the initiation of the expression of apical ectodermal ridge (AER)-specific genes, including fibroblast growth factor 8 (FGF8) and FGF4 occurred normally, the maintenance of these marker expressions was significantly defective in the hindlimb of Rspo2(-/-) mice. Consistent with the ligand role of R-spondins in the Wnt/beta-catenin signaling pathway, expression of Axin2 and Sp8, targets for beta-catenin signaling, within AER was greatly reduced in Rspo2(-/-) embryos. Furthermore, sonic hedgehog (Shh) signaling within the hindlimbs of Rspo2(-/-) mice was also significantly decreased. Rspo2 is expressed in the AER of all limb buds, however the stunted phenotype is significantly more severe in the hindlimbs than the forelimbs and strongly biased to the left side. Our findings strongly suggest that Rspo2 expression in the AER is required for AER maintenance likely by regulating Wnt/beta-catenin signaling.

Systematic bias in predictions of new drugs' budget impact: analysis of a sample of recent US drug launches.

OBJECTIVE: Expectations about the budget impact of new drug launches may affect payer behavior and ultimately consumer costs. Therefore, we evaluated the accuracy of pre-launch US budget impact estimates for a sample of new drugs. METHODS: We searched for publicly available budget impact estimates made pre-launch for drugs approved in the US from 1 September 2010 to 1 September 2015 and compared them to actual sales. Accuracy was calculated as the ratio of pre-launch estimate to actual sales. Quantitative analyses, including multivariate regressions, were used to identify factors associated with accuracy. RESULTS: We identified 25 budget impact estimates: 23 for one of 14 individual drugs and 2 for the category of PCSK9 inhibitors. The ratios of predicted to actual budget impact ranged from 0.2 (estimate was 20% of sales) for secukinumab to 37.5 (estimate was 37.5 × sales) for PCSK9 inhibitors. Mean ratio was 5.5. In multivariate analyses, larger eligible population, more recent estimate year (e.g. 2015 vs. 2012), and being first in class, were associated with statistically significant, greater overestimation of budget impact. CONCLUSIONS: For every $5.5 of predicted cost, there was $1 of actual cost to the healthcare system. This study, although based on a small, non-random sample, suggests possible cognitive bias on the part of the estimators. Overestimating budget impact may lead to early access restrictions, higher copays, and other changes that ultimately impact patients. Analysts and non-profits should be attuned to likely sources of error in order to improve their predictions.

Measuring the Value of New Drugs: Validity and Reliability of 4 Value Assessment Frameworks in the Oncology Setting.

BACKGROUND: Several organizations have developed frameworks to systematically assess the value of new drugs. OBJECTIVE: To evaluate the convergent validity and interrater reliability of 4 value frameworks to understand the extent to which these tools can facilitate value-based treatment decisions in oncology. METHODS: Eight panelists used the American Society of Clinical Oncology (ASCO), European Society for Medical Oncology (ESMO), Institute for Clinical and Economic Review (ICER), and National Comprehensive Cancer Network (NCCN) frameworks to conduct value assessments of 15 drugs for advanced lung and breast cancers and castration-refractory prostate cancer. Panelists received instructions and published clinical data required to complete the assessments, assigning each drug a numeric or letter score. Kendall's Coefficient of Concordance for Ranks (Kendall's W) was used to measure convergent validity by cancer type among the 4 frameworks. Intraclass correlation coefficients (ICCs) were used to measure interrater reliability for each framework across cancers. Panelists were surveyed on their experiences. RESULTS: Kendall's W across all 4 frameworks for breast, lung, and prostate cancer drugs was 0.560 (P= 0.010), 0.562 (P = 0.010), and 0.920 (P < 0.001), respectively. Pairwise, Kendall's W for breast cancer drugs was highest for ESMO-ICER and ICER-NCCN (W = 0.950, P = 0.019 for both pairs) and lowest for ASCO-NCCN (W = 0.300, P = 0.748). For lung cancer drugs, W was highest pairwise for ESMO-ICER (W = 0.974, P = 0.007) and lowest for ASCO-NCCN (W = 0.218, P = 0.839); for prostate cancer drugs, pairwise W was highest for ICER-NCCN (W = 1.000, P < 0.001) and lowest for ESMO-ICER and ESMO-NCCN (W = 0.900, P = 0.052 for both pairs). When ranking drugs on distinct framework subdomains, Kendall's W among breast cancer drugs was highest for certainty (ICER, NCCN: W = 0.908, P = 0.046) and lowest for clinical benefit (ASCO, ESMO, NCCN: W = 0.345, P = 0.436). Among lung cancer drugs, W was highest for toxicity (ASCO, ESMO, NCCN: W = 0. 944, P < 0.001) and lowest for certainty (ICER, NCCN: W = 0.230, P = 0.827); and among prostate cancer drugs, it was highest for quality of life (ASCO, ESMO: W = 0.986, P = 0.003) and lowest for toxicity (ASCO, ESMO, NCCN: W = 0.200, P = 0.711). ICC (95% CI) for ASCO, ESMO, ICER, and NCCN were 0.800 (0.660-0.913), 0.818 (0.686-0.921), 0.652 (0.466-0.834), and 0.153 (0.045-0.371), respectively. When scores were rescaled to 0-100, NCCN provided the narrowest band of scores. When asked about their experiences using the ASCO, ESMO, ICER, and NCCN frameworks, panelists generally agreed that the frameworks were logically organized and reasonably easy to use, with NCCN rated somewhat easier. CONCLUSIONS: Convergent validity among the ASCO, ESMO, ICER, and NCCN frameworks was fair to excellent, increasing with clinical benefit subdomain concordance and simplicity of drug trial data. Interrater reliability, highest for ASCO and ESMO, improved with clarity of instructions and specificity of score definitions. Continued use, analyses, and refinements of these frameworks will bring us closer to the ultimate goal of using value-based treatment decisions to improve patient care and outcomes. DISCLOSURES: This work was funded by Eisai Inc. Copher and Knoth are employees of Eisai Inc. Bentley, Lee, Zambrano, and Broder are employees of Partnership for Health Analytic Research, a health services research company paid by Eisai Inc. to conduct this research. For this study, Cohen, Huynh, and Neville report fees from Partnership for Health Analytic Research. Outside of this study, Cohen receives grants and direct consulting fees from various companies that manufacture and market pharmaceuticals. Mei reports a grant from Eisai Inc. during this study. The other authors have no disclosures to report. Study concept and design were contributed by Bentley and Broder, with assistance from Elkin and Cohen. Bentley took the lead in data collection, along with Elkin, Huynh, Mukherjea, Neville, Mei, Popescu, Lee, and Zambrano. Data interpretation was performed by Bentley and Broder, along with Elkin, Cohen, Copher, and Knoth. The manuscript was written primarily by Bentley, along with Elkin and Broder, and revised by Bentley, Broder, Elkin, Cohen, Copher, and Knoth. Select components of this work's methods were presented at ISPOR 19th Annual European Congress held in Vienna, Austria, October 29-November 2, 2016, and Society for Medical Decision Making 38th Annual North American Meeting held in Vancouver, Canada, October 23-26, 2016.

Frequency of apnea, bradycardia, and desaturations following first diphtheria-tetanus-pertussis-inactivated polio-Haemophilus influenzae type B immunization in hospitalized preterm infants.

BACKGROUND: Adverse cardiorespiratory events including apnea, bradycardia, and desaturations have been described following administration of the first diphtheria-tetanus-pertussis-inactivated polio-Haemophilus influenzae type B (DTP-IPV-Hib) immunization to preterm infants. The effect of the recent substitution of acellular pertussis vaccine for whole cell pertussis vaccine on the frequency of these events requires further study. METHODS: Infants with gestational age of < or = 32 weeks who received their first DTP-IPV-Hib immunization prior to discharge from two Edmonton Neonatal Intensive Care Units January 1, 1996 to November 30, 2000 were eligible for the study. Each immunized infant was matched by gestational age to one control infant. The number of episodes of apnea, bradycardia, and/or desaturations (ABD) and the treatment required for these episodes in the 72 hours prior to and 72 hours post-immunization (for the immunized cohort) or at the same post-natal age (for controls) was recorded. RESULTS: Thirty-four infants who received DTP-IPV-Hib with whole cell pertussis vaccine, 90 infants who received DTP-IPV-Hib with acellular pertussis vaccine, and 124 control infants were entered in the study. Fifty-six immunized infants (45.1%) and 36 control infants (29.0%) had a resurgence of or increased ABD in the 72 hours post-immunization in the immunized infants and at the same post-natal age in the controls with an adjusted odds ratio for immunized infants of 2.41 (95% CI 1.29,4.51) as compared to control infants. The incidence of an increase in adverse cardiorespiratory events post-immunization was the same in infants receiving whole cell or acellular pertussis vaccine (44.1% versus 45.6%). Eighteen immunized infants (14.5%) and 51 control infants (41.1%) had a reduction in ABD in the 72 hours post- immunization or at the equivalent postnatal age in controls for an odds ratio of 0.175 (95%CI 0.08, 0.39). The need for therapy of ABD in the immunized infants was not statistically different from the control infants. Lower weight at the time of immunization was a risk factor for a resurgence of or increased ABD post-immunization. Birth weight, gestational age, postnatal age or sex were not risk factors. CONCLUSION: There is an increase in adverse cardiorespiratory events following the first dose of DTP-IPV-Hib in preterm infants. Lower current weight was identified as a risk factor, with the risk being equivalent for whole cell versus acellular pertussis vaccine. Although most of these events are of limited clinical significance, cardiorespiratory monitoring of infants who are sufficiently preterm that they are receiving their first immunization prior to hospital discharge should be considered for 72 hours post-immunization.

ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets.

Proteins in the B cell CLL/lymphoma 2 (BCL-2) family are key regulators of the apoptotic process. This family comprises proapoptotic and prosurvival proteins, and shifting the balance toward the latter is an established mechanism whereby cancer cells evade apoptosis. The therapeutic potential of directly inhibiting prosurvival proteins was unveiled with the development of navitoclax, a selective inhibitor of both BCL-2 and BCL-2-like 1 (BCL-X(L)), which has shown clinical efficacy in some BCL-2-dependent hematological cancers. However, concomitant on-target thrombocytopenia caused by BCL-X(L) inhibition limits the efficacy achievable with this agent. Here we report the re-engineering of navitoclax to create a highly potent, orally bioavailable and BCL-2-selective inhibitor, ABT-199. This compound inhibits the growth of BCL-2-dependent tumors in vivo and spares human platelets. A single dose of ABT-199 in three patients with refractory chronic lymphocytic leukemia resulted in tumor lysis within 24 h. These data indicate that selective pharmacological inhibition of BCL-2 shows promise for the treatment of BCL-2-dependent hematological cancers.

Differential transcriptional activation of matrix metalloproteinase-2 and membrane type-1 matrix metalloproteinase by experimental deep venous thrombosis and thrombin.

OBJECTIVE: Resolution of deep venous thrombosis (DVT) is involved in the pathogenesis of postthrombotic syndrome. Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that are critical in angiogenesis and tissue remodeling. We hypothesized that MMP-2 and its membrane-bound activator membrane type-1 matrix metalloproteinase (MT1-MMP) expression would be expressed and activated during the resolution of DVT. METHODS: DVT was generated by caval ligation in wild-type and MMP-2 transgenic reporter mice. Ligated and sham-operated (control) cavae were analyzed for MMP-2 transcription (beta-galactosidase activity in MMP-2 reporter mice) and MT1-MMP mRNA by real-time polymerase chain reaction. MMP-2 activity was determined by zymography, and immunohistochemical staining for beta-galactosidase and MT1-MMP protein was used to localize expression. Human umbilical vascular endothelial cells (HUVEC) were treated with 10 U/mL thrombin and MMP-2 and MT1-MMP mRNA levels and MMP-2 activity was determined. RESULTS: MMP-2 activity increased 71% (n = 5, P < .05) at day 8 in ligated vs control cavae by zymography. beta-galactosidase activity showed a 1.2-fold (n = 8, P < .05) and 1.7-fold (n = 8, P < .05) induction in MMP-2 transcription at day 3 and day 8, respectively. No significant MT1-MMP gene induction was seen at day 3 in ligated vs control cavae, but MT1-MMP mRNA was upregulated 2.5-fold (n = 8, P < .05) in ligated cavae at day 8. Immunohistochemical staining localized MMP-2 and MT1-MMP expression to the vein wall and cellular infiltrates of the thrombus. Thrombin-treated HUVEC showed differential responses of MMP-2 and MT1-MMP. Zymography of conditioned media and cell lysates illustrated a 220% (152.6 +/- 8.6 vs 69.445 +/- 5.46 pixels/unit area, n = 5, P < .05) and 150% (74.1 +/- 7.3 vs 49.2 +/- 5.7 pixels/unit area, n = 5, P < .05) increases in MMP-2 activity respectively. MMP-2 mRNA levels were downregulated 30% (0.48 +/- 0.023 vs 0.63 +/- 0.035 copies of MMP-2 mRNA/copy GAPDH, n = 5, P < .05), whereas MT1-MMP message was upregulated 250% (0.147 +/- 0.009 vs 0.059 +/- 0.005 copies of MT1-MMP mRNA/copy GAPDH, n = 5, P < .05). CONCLUSIONS: Resolution of DVT is associated with increased MMP-2 transcription and activity as well as MT1-MMP expression. Thrombin may mediate the increase in MT1-MMP noted in DVT. This is the first article studying MMP-2 and MT1-MMP transcription in DVT. These findings add DVT resolution to the class of inflammatory and fibrotic disorders in which transcriptional activation of the MT1-MMP/MMP-2 genes occurs and identify a potential therapeutic target to modulate this clinically relevant process. CLINICAL RELEVANCE: Postthrombotic syndrome remains a significant clinical problem after deep venous thrombosis (DVT), but the cellular and molecular mechanisms involved in thrombus resolution and vein wall fibrosis remain undefined. Matrix metalloproteinase (MMP) enzymes are critical to cell migration and matrix breakdown. We identify gene transcription and activity of two MMP isoforms, MMP-2 and MMP-14 (membrane type MMP 1, MT1-MMP) in the resolution phase of experimental DVT and in thrombin-treated endothelial cells. These studies define new proteases potentially important to resolution of DVT and development of postthrombotic syndrome.

Intronic regulation of matrix metalloproteinase-2 revealed by in vivo transcriptional analysis in ischemia.

Matrix metalloproteinase-2 (MMP-2) plays an essential role in angiogenesis and arteriogenesis, two processes critical to restoration of tissue perfusion after ischemia. MMP-2 expression is increased in tissue ischemia, but the responsible mechanisms remain unknown. We studied the transcriptional activation of the MMP-2 gene in a model of hindlimb ischemia by using various MMP-2-lacZ reporter mice and chromatin immunoprecipitation. MMP-2 activity and mRNA were increased after hindlimb ischemia. Mice with targeted deletion of MMP-2 had impaired restoration of perfusion and a high incidence of limb gangrene, indicating that MMP-2 plays a critical role in ischemia-induced revascularization. Ischemia induced the expression and binding of c-Fos, c-Jun, JunB, FosB, and Fra2 to a noncanonical activating protein-1 (AP-1) site present in the MMP-2 promoter and decreased binding of the transcriptional repressor JunD. Ischemia also activated the expression and binding of p53 to an adjacent enhancer site (RE-1) and increased expression and binding of nuclear factor of activated T-cells-c2 to consensus sequences within the first intron. Deletion of either the 5' AP-1/RE-1 region of the promoter or substitution of the first intron abolished ischemia-induced MMP-2 transcription in vivo. Thus, AP-1 transcription factors and intronic activation by nuclear factor of activated T-cells-c2 act in concert to drive ischemia-induced MMP-2 transcription. These findings define a critical role for MMP-2 in ischemia-induced revascularization and identify both previously uncharacterized regulatory elements within the MMP-2 gene and the cognate transcription factors required for MMP-2 activation in vivo after tissue ischemia.