Pulmonary Hypertension and Anastrozole (PHANTOM): A Randomized, Double-Blind, Placebo-Controlled Trial.

RATIONALE: Inhibition of aromatase with anastrozole reduces pulmonary hypertension in experimental models. OBJECTIVES: We aimed to determine whether anastrozole improved six-minute walk distance (6MWD) at six months in pulmonary arterial hypertension (PAH). METHODS: We performed a randomized, double-blind, placebo-controlled Phase II clinical trial of anastrozole in subjects with PAH at seven centers. Eighty-four post-menopausal women and men with PAH were randomized in a 1:1 ratio to receive anastrozole 1 mg or placebo by mouth daily, stratified by sex using permuted blocks of variable sizes. All subjects and study staff were masked. The primary outcome was the change from baseline in 6MWD at six months. Using intent-to-treat analysis, we estimated the treatment effect of anastrozole using linear regression models adjusted for sex and baseline 6MWD. Assuming 10% loss to follow-up, we anticipated having 80% power to detect a difference in the change in 6MWD of 22 meters. MEASUREMENTS AND MAIN RESULTS: Forty-one subjects were randomized to placebo and 43 to anastrozole and all received the allocated treatment. Three subjects in the placebo group and two in the anastrozole group discontinued study drug. There was no significant difference in the change in 6MWD at six months (placebo-corrected treatment effect -7.9 m, 95%CI -32.7 - 16.9, p = 0.53). There was no difference in adverse events between the groups. CONCLUSIONS: Anastrozole did not show a significant effect on 6MWD compared to placebo in post-menopausal women and men with PAH. Anastrozole was safe and did not show adverse effects. Clinical trial registration available at www. CLINICALTRIALS: gov, ID: NCT03229499.

Pulmonary arterial hypertension: Sex matters.

Pulmonary arterial hypertension (PAH) is a complex disease of multifactorial origin. While registries have demonstrated that women are more susceptible to the disease, females with PAH have superior right ventricle (RV) function and a better prognosis than their male counterparts, a phenomenon referred to as the 'estrogen paradox'. Numerous pre-clinical studies have investigated the involvement of sex hormones in PAH pathobiology, often with conflicting results. However, recent advances suggest that abnormal estrogen synthesis, metabolism and signalling underpin the sexual dimorphism of this disease. Other sex hormones, such as progesterone, testosterone and dehydroepiandrosterone may also play a role. Several non-hormonal factor including sex chromosomes and epigenetics have also been implicated. Though the underlying pathophysiological mechanisms are complex, several compounds that modulate sex hormones levels and signalling are under investigation in PAH patients. Further elucidation of the estrogen paradox will set the stage for the identification of additional therapeutic targets for this disease.

Data for analysis of catechol estrogen metabolites in human plasma by liquid chromatography tandem mass spectrometry.

Analysis of catechol estrogens (2 & 4 hydroxy-estrone and estradiol) has proven troublesome by liquid chromatography tandem mass spectrometry due to their low concentrations, short half-lives and temperature-labile nature. Derivatization to methyl piperazine analogues has been reported for a panel of 9 estrogens in, "Derivatization enhances analysis of estrogens and their bioactive metabolites in human plasma by liquid chromatography tandem mass spectrometry" (Denver et al., 2019). Data show alteration of the base catalyst in this method was required to allow detection of catechol estrogens to low levels. Data also highlight the challenges faced in chromatographic separation of isomers and isotopologues, which were partially overcome by employing an extended column length and reduced oven temperature. In addition, data analysis displayed significant matrix effects during quantitation in plasma, following solid-phase extraction, despite efficient recoveries.

Derivatization enhances analysis of estrogens and their bioactive metabolites in human plasma by liquid chromatography tandem mass spectrometry.

Estrogens regulate many diverse biological processes in health and disease. They circulate at a wide range of concentrations in females generating several active metabolites (hydroxy and methoxyestrogens). The metabolites are assumed to be present in much lower levels and are thought to contribute to diseases such as pulmonary arterial hypertension (PAH). Estrogen metabolites are challenging to quantify in plasma and currently available immunoassays are non-specific. Here we have developed and validated a novel assay to simultaneously quantify parent estrogens and their metabolites by mass spectrometry (MS). Estrogens were extracted from human plasma using solid phase extraction and derivatized using 1-(5-fluoro-2, 4-dinitrophenyl)-4-methylpiperazine (PPZ) before quaternization by methylation ("MPPZ"). MPPZ derivatives were separated and quantified by liquid chromatography tandem MS (LC-MS/MS) in positive electrospray ionization mode, using a QTrap 6500 + coupled to a Shimadzu Nexera X2. Separation was achieved using an ACE Excel 2 C18-PFP column (2 µm, 2.1 mm × 150 mm). The limits of quantification (LOQ) were 0.43-2.17 pg on column with a linear range from 2 or 10 - 2000 pg mL-1. Intra and inter-day precision and accuracy were acceptable (<20% at LOQ and <15% above). These derivatives demonstrated minimal degradation upon short-term storage at 15 °C (<20%) and longer term at -20 °C (<20%). Using this approach, estrone (E1) and estradiol (E2) were detected in plasma (0.5 mL) from healthy women and those with PAH but downstream metabolites 16-hydroxy-E1, 16-hydroxy-E2, 2-methoxy-E1 and 4-methoxy-E1 were only detected in plasma from diseased patients. These findings will next be tested robustly in large patient cohorts. This novel LC-MS/MS analysis of estrogens and their bioactive metabolites, using MPPZ derivatization, opens doors for the simultaneous analysis of a panel of estrogens in human plasma, across the endogenous range of concentrations encountered in health and disease.

Breast cancer cell cyclooxygenase-2 expression alters extracellular matrix structure and function and numbers of cancer associated fibroblasts.

Cyclooxygenase-2 (COX-2) is a critically important mediator of inflammation that significantly influences tumor angiogenesis, invasion, and metastasis. We investigated the role of COX-2 expressed by triple negative breast cancer cells in altering the structure and function of the extracellular matrix (ECM). COX-2 downregulation effects on ECM structure and function were investigated using magnetic resonance imaging (MRI) and second harmonic generation (SHG) microscopy of tumors derived from triple negative MDA-MB-231 breast cancer cells, and a derived clone stably expressing a short hairpin (shRNA) molecule downregulating COX-2. MRI of albumin-GdDTPA was used to characterize macromolecular fluid transport in vivo and SHG microscopy was used to quantify collagen 1 (Col1) fiber morphology. COX-2 downregulation decreased Col1 fiber density and altered macromolecular fluid transport. Immunohistochemistry identified significantly fewer activated cancer associated fibroblasts (CAFs) in low COX-2 expressing tumors. Metastatic lung nodules established by COX-2 downregulated cells were infrequent, smaller, and contained fewer Col1 fibers.COX-2 overexpression studies were performed with tumors derived from triple negative SUM-149 breast cancer cells lentivirally transduced to overexpress COX-2. SHG microscopy identified significantly higher Col1 fiber density in COX-2 overexpressing tumors with an increase of CAFs. These data expand upon the roles of COX-2 in shaping the structure and function of the ECM in primary and metastatic tumors, and identify the potential role of COX-2 in modifying the number of CAFs in tumors that may have contributed to the altered ECM.

The use of Bioptron light (polarized, polychromatic, non-coherent) therapy for the treatment of acute ankle sprains.

Purpose The purpose of this study was to investigate the efficacy of Bioptron light therapy for the treatment of acute ankle sprains. Method A parallel group, single-blind, controlled study was carried out in patients with grade II acute ankle sprains. Patients were randomly allocated into two treatment groups (n = 25 for each). Both groups received cryotherapy, and the test group also received Bioptron light therapy. All treatments were performed daily for 5 d. Evaluations included self-reported pain via a visual analogue scale, degree of ankle edema, and ankle range of motion via goniometry carried out before the treatment and at the end of the treatment. Results The test group showed the largest magnitude of improvement for all evaluations at treatment five, and the between-group differences observed were statistically significant (p < 0.0005 for each). Conclusions These data provide preliminary evidence of the efficacy of Bioptron light therapy supplemented with cryotherapy for the treatment of acute ankle sprains; however, larger studies are required to confirm these results. Implications for Rehabilitation Ankle sprains are common acute injuries among professional and recreational sports players but also among people in general. Cryotherapy is the first-standard treatment of acute ankle sprains. Phototherapy such as Bioptron light has been recommended supplement to cryotherapy to reduce the symptoms of ankle sprains. The results of the present trial showed that using BIOPTRON LIGHT and cryotherapy the rehabilitation period of acute ankle sprains can be reduced.

Comparison of effects of eccentric training, eccentric-concentric training, and eccentric-concentric training combined with isometric contraction in the treatment of lateral elbow tendinopathy.

STUDY DESIGN: RCT. INTRODUCTION: Lateral elbow tendinopathy is a common clinical condition. eccentric exercises. eccentric-concentric loading and, isometric exercises are indicated to reduce and manage tendon pain. PURPOSE OF THE STUDY: To compare the effectiveness of eccentric training, eccentric-concentric training, and eccentric-concentric training combined with isometric contraction in the treatment of lateral elbow tendinopathy. METHODS: A randomized clinical trial was carried out in a rheumatology and rehabilitation center. A group of 34 patients with later elbow tendinopathy was randomly allocated to Group A (n = 11) who underwent eccentric training, Group B had eccentric-concentric and Group C who had eccentric-concentric and isometric training. All patients received 5 treatments per week for 4 weeks. Pain was evaluated using a visual analog scale and function using a visual analog scale and pain-free grip strength at the end of the 4-week course of treatment (week 4) and 1 month (week 8) after the end of treatment. RESULTS: The eccentric-concentric training combined with isomentric contractions produced the largest effect in the reduction of pain and improvement of function at the end of the treatment (P < .05) and at any of the follow-up time points (P < .05). CONCLUSION: The eccentric-concentric training combined with isomentric contractions was the most effective treatment. Future well-designed studies are needed to confirm the results of the present trial. LEVEL OF EVIDENCE: III.

COX-2 dependent regulation of mechanotransduction in human breast cancer cells.

The ability of living cells to exert physical forces upon their surrounding is a necessary prerequisite for diverse biological processes, such as local cellular migrations in wound healing to metastatic-invasion of cancer. How forces are coopted in metastasis has remained unclear, however, because the mechanical interplay between cancer cells and the various stromal components has not been experimentally accessible. Current dogma implicates inflammation in these mechanical processes. Using Fourier transform traction microscopy, we measured the force-generating capacity of human breast cancer cells occupying a spectrum of invasiveness as well as basal and inducible COX-2 expression (MCF-7

COX-2 in cancer: Gordian knot or Achilles heel?

The networks of blood and lymphatic vessels and of the extracellular matrix and their cellular and structural components, that are collectively termed the tumor microenvironment, are frequently co-opted and shaped by cancer cells to survive, invade, and form distant metastasis. With an enviable capacity to adapt to continually changing environments, cancer represents the epitome of functional chaos, a stark contrast to the hierarchical and organized differentiation processes that dictate the development and life of biological organisms. The consequences of changing landscapes such as hypoxia and acidic extracellular pH in and around tumors create a cascade of changes in multiple pathways and networks that become apparent only several years later as recurrence and metastasis. These molecular and phenotypic changes, several of which are mediated by COX-2, approach the complexities of a "Gordian Knot." We review evidence from our studies and from literature suggesting that cyclooxygenase-2 (COX-2) biology presents a nodal point in cancer biology and an "Achilles heel" of COX-2-dependent tumors.

Wireless electrical stimulation: an innovative powerful tool for the treatment of a complicated chronic ulcer.

High-voltage electrical stimulation has been long proposed as a method of accelerating the wound healing process. Its beneficial effect has been successfully evaluated in the treatment of a number of chronic ulcers and burns. We present here the implementation of a new wireless electrical stimulation technique for the treatment of a complicated chronic ulcer of the lower limb. The device is transferring charges to the wound, without any contact with it, creating a microcurrent that is able to generate the current of injury. The results suggest that this easy-to-use method is an effective therapeutic option for chronic ulcers.

Prostaglandin E2 promotes the nuclear accumulation of lymphoid enhancer factor-1 in poorly differentiated breast cancer cells.

Products of the COX reaction are frequently elevated in solid tumors and their roles in the malignant phenotype have been extensively investigated. We have shown that COX-2 is essential for the growth of MDA-MB-231 cells in the fat pad of SCID mice and for their extrapulmonary colonization following injection in the tail vein of SCID mice. The molecular changes that follow shRNA-mediated silencing of COX-2 include a significant downregulation of LEF-1, a transcription factor normally activated during development following the Wnt-induced nuclear translocation of ß-catenin. We also report that COX-2-silenced cells have reduced nuclear accumulation of LEF-1 protein and that the COX-2 product PGE(2) partially restored nuclear LEF-1 expression in COX-2-silenced cells. Further, we demonstrate that, like parental COX-2 containing MDA-MB-231 cells, COX-2-silenced cells maintain nuclear localization of ß-catenin.

Noninvasive imaging identifies new roles for cyclooxygenase-2 in choline and lipid metabolism of human breast cancer cells.

The expression of cyclooxygenase-2 (COX-2) is observed in approximately 40% of breast cancers. A major product of the COX-2-catalyzed reaction, prostaglandin E(2), is an inflammatory mediator that participates in several biological processes, and influences invasion, vascularization and metastasis. Using noninvasive MRI and MRS, we determined the effect of COX-2 downregulation on the metabolism and invasion of intact poorly differentiated MDA-MB-231 human breast cancer cells stably expressing COX-2 short hairpin RNA. Dynamic tracking of invasion, extracellular matrix degradation and metabolism was performed with an MRI- and MRS-compatible cell perfusion assay under controlled conditions of pH, temperature and oxygenation over the course of 48 h. COX-2-silenced cells exhibited a significant decrease in invasion relative to parental cells that was consistent with the reduced expression of invasion-associated matrix metalloproteinase genes and an increased level of the tissue inhibitor of metalloproteinase-1. We identified, for the first time, a role for COX-2 in mediating changes in choline phospholipid metabolism, and established that choline kinase expression is partly dependent on COX-2 function. COX-2 silencing resulted in a significant decrease in phosphocholine and total choline that was detected by MRS. In addition, a significant increase in lipids, as well as lipid droplet formation, was observed. COX-2 silencing transformed parental cell metabolite patterns to those characteristic of less aggressive cancer cells. These new functional roles of COX-2 may identify new biomarkers and new targets for use in combination with COX-2 targeting to prevent invasion and metastasis.

Exploiting the tumor microenvironment for theranostic imaging.

The integration of chemistry and molecular biology with imaging is providing some of the most exciting opportunities in the treatment of cancer. The field of theranostic imaging, where diagnosis is combined with therapy, is particularly suitable for a disease as complex as cancer, especially now that genomic and proteomic profiling can provide an extensive 'fingerprint' of each tumor. Using this information, theranostic agents can be shaped for personalized treatment to target specific compartments, such as the tumor microenvironment (TME), whilst minimizing damage to normal tissue. These theranostic agents can also be used to target multiple pathways or networks by incorporating multiple small interfering RNAs (siRNAs) within a single agent. A decade ago genetic alterations were the primary focus in cancer research. Now it is apparent that the tumor physiological microenvironment, interactions between cancer cells and stromal cells, such as endothelial cells, fibroblasts and macrophages, the extracellular matrix (ECM), and a host of secreted factors and cytokines, influence progression to metastatic disease, aggressiveness and the response of the disease to treatment. In this review, we outline some of the characteristics of the TME, describe the theranostic agents currently available to target the TME and discuss the unique opportunities the TME provides for the design of novel theranostic agents for cancer therapy.

Hypoxic tumor microenvironments reduce collagen I fiber density.

Although the mechanisms through which hypoxia influences several phenotypic characteristics such as angiogenesis, selection for resistance to apoptosis, resistance to radiation and chemotherapy, and increased invasion and metastasis are well characterized, the relationship between tumor hypoxia and components of the extracellular matrix (ECM) is relatively unexplored. The collagen I (Col1) fiber matrix of solid tumors is the major structural part of the ECM. Col1 fiber density can increase tumor initiation, progression, and metastasis, with cancer cell invasion occurring along radially aligned Col1 fibers. Here we have investigated the influence of hypoxia on Col1 fiber density in solid breast and prostate tumor models. Second harmonic generation (SHG) microscopy was used to detect differences in Col1 fiber density and volume between hypoxic and normoxic tumor regions. Hypoxic regions were detected by fluorescence microscopy, using tumors derived from human breast and prostate cancer cell lines stably expressing enhanced green fluorescent protein (EGFP) under transcriptional control of the hypoxia response element. In-house fiber analysis software was used to quantitatively analyze Col1 fiber density and volume from the SHG microscopy images. Normoxic tumor regions exhibited a dense mesh of Col1 fibers. In contrast, fewer and structurally altered Col1 fibers were detected in hypoxic EGFP-expressing tumor regions. Microarray gene expression analyses identified increased expression of lysyl oxidase and reduced expression of some matrix metalloproteases in hypoxic compared with normoxic cancer cells. These results suggest that hypoxia mediates Col1 fiber restructuring in tumors, which may impact delivery of macromolecular agents as well as dissemination of cells.

Molecular and functional imaging of invasion and metastasis: windows into the metastatic cascade.

The ability of cancer cells to invade, metastasize, and form distant colonies, is one of the key characteristics that confers lethality to cancer. Metastatic cancer cells typically become refractory to treatment. The metastatic cascade is a multi-step process that is governed by events within the cancer cell, the tumor microenvironment, and the distant environments that are invaded and colonized by the cancer cells. Noninvasive imaging techniques are facilitating a close examination of the stepwise journey of the cancer cell from the primary tumor to the distant metastatic site. Here we have discussed the metastatic process, and how molecular and functional imaging of cancer are providing new insights into the metastatic cascade that can be exploited for treatment of metastatic disease.

Comparing the effects of exercise program and low-level laser therapy with exercise program and polarized polychromatic non-coherent light (bioptron light) on the treatment of lateral elbow tendinopathy.

BACKGROUND DATA: The use of low-level laser therapy (LLLT) and polarized polychromatic non-coherent light as supplements to an exercise program has been recommended for the management of lateral elbow tendinopathy (LET). OBJECTIVE: To investigate whether an exercise program supplemented with LLLT is more successful than an exercise program supplemented with polarized polychromatic non-coherent light in treating LET. MATERIALS AND METHODS: Patients with unilateral LET for at least 4 wk were sequentially allocated to receive either an exercise program with LLLT or an exercise program with polarized polychromatic non-coherent light. The exercise program consisted of eccentric and static stretching exercises of wrist extensors. In the LLLT group a 904-nm Ga-As laser was used in continuous mode, and the power density was 130 mW/cm(2), and the dose was 0.585 J/point. In the group receiving polarized polychromatic non-coherent light the Bioptron 2 was used to administer the dose perpendicularly to the lateral epicondyle at three points at an operating distance of 5-10 cm for 6 min at each position. The outcome measures were pain and function and were evaluated at baseline, at the end of the treatment (week 4), and 3 mo after the end of treatment (week 16). RESULTS: Fifty patients met the inclusion criteria. At the end of treatment there was a decline in pain and a rise in function in both groups compared with baseline (p < 0.0005 on the paired t-test). There were no significant differences in the reduction of pain and the improvement of function between the groups at the end of treatment and at the 3-mo follow-up (p > 0.0005 on the independent t-test). CONCLUSIONS: The results suggest that the combination of an exercise program with LLLT or polarized polychromatic non-coherent light is an adequate treatment for patients with LET. Further research to establish the relative and absolute effectiveness of such a treatment approach is needed.

Inflammation, but not hypoxia, mediated HIF-1alpha activation depends on COX-2.

The COX pathway has been a target for pharmaceutical intervention in diseases with a high inflammatory component ranging from asthma and Alzheimer's to arthritis and cancer. A major transcriptional promoter of the malignant phenotype, HIF-1alpha, has been observed to be regulated by the COX-2 product PGE2. Here we show that HIF-1alpha protein significantly accumulated in human breast cancer MDA-MB-231 cells in response to the pro-inflammatory cytokine IL-1beta, but not in COX-2-silenced MDA-MB-231 cells. In contrast HIF-1alpha expression could be detected in COX-2- silenced cells in response to the hypoxia-mimetic agent CoCl(2) and hypoxia. Gene expression profiling in COX-2-containing and COX-2-silenced cells showed that the hypoxia-induced transcriptional response is largely unaffected by COX-2 silencing. These data suggest that the profound effects of COX-2 silencing on inhibiting invasion, tumor growth and metastasis from MDA-MB-231 cells are dependent on the induction of IL-1beta-dependent COX-2 and HIF-1alpha but are independent of hypoxia

The malignant phenotype of breast cancer cells is reduced by COX-2 silencing.

The cyclooxygenase (COX) pathway is currently targeted for therapeutic intervention in different cancers. We have previously shown that silencing of COX-2 in the poorly differentiated metastatic breast cell line MDA-MB-231 by RNA interference markedly delayed tumor onset and inhibited metastasis. To understand the functional effects of COX-2 silencing underlying the inhibition of tumor growth and metastasis previously reported, we investigated changes in these cells for a number of cancer-associated phenotypes. Cyclooxygenase-2-silenced cells were less able to acidify tissue culture medium, a response that could partly be attributed to decreased lactate production or export detected by reduced lactate in the medium. Consistent with the significantly reduced transcript levels of hyaluronan synthase 2, an enzyme responsible for the total level of hyaluronan secreted by these cells, COX-2 silencing resulted in lower hyaluronan levels secreted in culture medium. Inhibition of human umbilical vein endothelial cell network association in a coculture assay was also observed in COX-2-silenced cells. These data highlight the functional role of COX-2 in pathways that mediate increased malignancy.

Silencing of cyclooxygenase-2 inhibits metastasis and delays tumor onset of poorly differentiated metastatic breast cancer cells.

Cyclooxygenases (COX) are rate-limiting enzymes involved in the conversion of PLA(2)-mobilized arachidonic acid into prostaglandins and thromboxanes. COX-2 is a key mediator of inflammation during both physiologic and pathologic responses to endogenous stimuli and infectious agents. Its overexpression has been detected in different cancers, including that of the breast. Using RNA interference, we have reduced the expression of COX-2 in the highly malignant breast cancer cell line MDA-MB-231 below detectable levels in response to interleukin-1 beta or 12-O-tetradecanoylphorbol-13-acetate treatment. Microarray analysis showed that COX-2 silencing resulted in the loss of mRNA expression of several oncogenic markers, such as matrix metalloproteinase-1, chemokine (C-X-C motif) receptor 4, and interleukin-11, which have been correlated with poor disease outcome, and in the up-regulation of antimetastatic transcripts, such as thrombospondin-1 and Epstein-Barr-Induced 3. Cells lacking COX-2 were less able to invade reconstituted extracellular matrix than parental cells in vitro. Consistent with these changes, loss of COX-2 resulted in the abolition or the significant delay of tumor onset when the cells were injected in the mammary fat pad of severe combined immunodeficient mice. Finally, silencing of COX-2 resulted in the inhibition of metastasis to the lungs of severe combined immunodeficient mice after intravenous injection. These data show that silencing of COX-2 abolishes the metastatic potential of MDA-MB-231 cells in vivo.