Peak steps: Capacity for activity improves after adding approved therapy in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) patients have low activity. Activity intensity or duration could be a measure of clinical status or improvement. We aimed to determine whether standard or novel actigraphy measures could detect increases in activity after adding therapy. This was a prospective, single-center observational study evaluating activity after adding therapy in Group 1 PAH; we also report a validation cohort. For our study, two different accelerometers were used, a wrist (ActiGraph) and chest (MC10) device. Patients were analyzed in two groups, Treatment Intensification (TI, adding therapy) or Stable. Both groups had baseline monitoring periods of 7 days; the TI group had follow-up at 3 months, while Stables had follow-up within 4 weeks to assess stability. Activity time and steps were reported from both devices' proprietary algorithms. In ActiGraph only, steps in 1-min intervals throughout the day were ranked (not necessarily contiguous). Average values for each week were calculated and compared using nonparametric testing. Thirty patients had paired data (11 Stable and 19 TI). There was no between-group difference at baseline; we did not observe therapy-associated changes on average daily steps or activity time/intensity. The top 5 min of steps (capacity) increased after adding therapy; there was no difference in the stable group. This key finding was validated in a previously reported randomized trial studying a behavioral intervention to increase exercise. Total daily activity metrics are influenced by both disease and non-disease factors, making therapy-associated change difficult to detect. Peak minute steps were a treatment-responsive marker in both a pharmacologic and training intervention.

Cardiac Effort to Compare Clinic and Remote 6-Minute Walk Testing in Pulmonary Arterial Hypertension.

BACKGROUND: The SARS-CoV-2 pandemic has limited objective physiologic assessments. A standardized remote alternative is not currently available. "Cardiac effort" (CE), that is, the total number of heart beats divided by the 6-min walk test (6MWT) distance (beats/m), has improved reproducibility in the 6MWT and correlated with right ventricular function in pulmonary arterial hypertension. RESEARCH QUESTION: Can a chest-based accelerometer estimate 6MWT distance remotely? Is remote cardiac effort more reproducible than 6MWT distance when compared with clinic assessment? STUDY DESIGN AND METHODS: This was a single-center, prospective observational study, with institutional review board approval, completed between October 2020 and April 2021. Group 1 subjects with pulmonary arterial hypertension, receiving stable therapy for > 90 days, completed four to six total 6MWTs during a 2-week period to assess reproducibility. The first and last 6MWTs were performed in the clinic; two to four remote 6MWTs were completed at each participant's discretion. Masks were not worn. BioStamp nPoint sensors (MC10) were worn on the chest to measure heart rate and accelerometry. Two blinded readers counted laps, using accelerometry data obtained on the clinic or user-defined course. Averages of clinic variables and remote variables were used for Wilcoxon matched-pairs signed rank tests, Bland-Altman plots, or Spearman correlation coefficients. RESULTS: Estimated 6MWT distance, using the MC10, correlated strongly with directly measured 6MWT distance (r = 0.99; P < .0001; in 20 subjects). Remote 6MWT distances were shorter than clinic 6MWT distances: 405 m (330-464 m) vs 389 m (312-430 m) (P = .002). There was no difference between in-clinic and remote CE: 1.75 beats/m (1.48-2.20 beats/m) vs 1.86 beats/m (1.57-2.14 beats/m) (P = .14). INTERPRETATION: Remote 6MWT was feasible on a user-defined course; 6MWT distance was shorter than clinic distance. CE calculated by chest heart rate and accelerometer-estimated distance provides a reproducible remote assessment of exercise tolerance, comparable to the clinic-measured value.

Comparison of chest- and wrist-based actigraphy in pulmonary arterial hypertension.

Aims: Activity trackers for clinical trials and remote monitoring are appealing as they provide objective data outside of the clinic setting. Algorithms determine physical activity intensity and count steps. Multiple studies show physical inactivity in pulmonary arterial hypertension (PAH). There are no studies comparing different activity trackers worn on different parts of the body in PAH. We had patients with PAH simultaneously wear two different accelerometers, compared measures between the two devices, and correlated the measures with standard clinical metrics in PAH. Methods and results: This was a single-centre, prospective observational study. Daily physical activity and daily total steps were measured using Actigraph GT9X Link and MC10 Biostamp nPoint for 5-10 days. Actigraph was worn on the non-dominant hand and the MC10 Biostamp nPoint was worn on the chest and leg with disposable adhesives. Twenty-two participants wore both accelerometers >12 h/day for an average 7.8 days. The average activity time measured by Actigraph was significantly higher than that measured by MC10 (251 ± 25 min vs. 113 ± 18 min, P = 0.0001). Actigraph's algorithm reported more time in light activity than moderate (190 ± 62 min vs. 60 ± 56 min, P = 0.0001). REVEAL 2.0 scores correlated highly with activity time measured using either device. Invasively measured haemodynamics within 7 days did not correlate with activity time or daily steps. Conclusion: Different activity trackers yield discordant results in PAH patients. Further studies are needed in determining the best device, optimal wear time, and different thresholds for activities in chronic diseases.

Heart rate monitoring improves clinical assessment during 6-min walk.

Right ventricular (RV) function is a predictor of outcomes in pulmonary arterial hypertension (PAH). The 6-min walk test (6MWT) is likely an indirect measure of RV function during exercise, but changes in absolute walk distance can also be influenced by factors like effort and musculoskeletal disease. Paired 6MWT with continuous electrocardiogram monitoring was performed in stable PAH patients, patients adding PAH therapies, and healthy controls. Heart rate expenditure (HRE) was calculated (integrating pulse during 6MWT) and then divided by walk distance (HRE/d). We also evaluated changes in peak heart rate, time above age-adjusted maximum predicted heart rate, and heart rate at 6 min. HRE/d was compared to invasive hemodynamic measures in patients who had right heart catheterization performed within seven days, WHO functional class assessment, and Emphasis 10 questionnaire. We measured two 6MWT in 15 stable PAH patients, 13 treatment intensification patients, and 8 healthy controls. HRE/d was reproducible in the stable PAH group (median difference, -0.79%), while it decreased (median difference, 23%, p = 0.0001) after adding vasodilator therapy. In 11 patients with right heart catheterization, HRE/d correlated strongly with stroke volume, r = -0.72, p = 0.01. Peak heart rate decreased after adding vasodilator therapy. HRE/d also correlated with WHO functional class and Emphasis 10 score. Continuous heart rate monitoring during 6MWT provides valuable physiologic data accounting for effort. HRE/d appears to enhance test reproducibility in stable patients while detecting change after adding therapy as compared to walk distance alone.

Estrogen maintains myometrial tumors in a lymphangioleiomyomatosis model.

Lymphangioleiomyomatosis (LAM) is a rare disease in women. Patients with LAM develop metastatic smooth-muscle cell adenomas within the lungs, resulting in reduced pulmonary function. LAM cells contain mutations in tuberous sclerosis genes (TSC1 or TSC2), leading to up-regulation of mTORC1 activity and elevated proliferation. The origin of LAM cells remains unknown; however, inactivation of Tsc2 gene in the mouse uterus resulted in myometrial tumors exhibiting LAM features, and approximately 50% of animals developed metastatic myometrial lung tumors. This suggests that LAM tumors might originate from the uterine myometrium, possibly explaining the overwhelming prevalence of LAM in female. Here, we demonstrate that mouse Tsc2-null myometrial tumors exhibit nearly all the features of LAM, including mTORC1/S6K activation, as well as expression of melanocytic markers and matrix metalloproteinases (MMPs). Estrogen ablation reduces S6K signaling and results in Tsc2-null myometrial tumor regression. Thus, even without TSC2, estradiol is required to maintain tumors and mTORC1/S6K signaling. Additionally, we find that MMP-2 and -9, as well as neutrophil elastase (NE), are overexpressed in Tsc2-null myometrial tumors in an estrogen-dependent fashion. In vivo fluorescent imaging using MMP- or NE-sensitive optical biomarkers confirms that protease activity is specific to myometrial tumors. Similar to LAM cells, uterine Tsc2-null myometrial cells also overexpress melanocytic markers in an estrogen-dependent fashion. Finally, we identify glycoprotein NMB (GPNMB) as a melanocytic marker up-regulated in Tsc2-null mouse uteri and human LAM samples. Our data highlight the potential importance of estradiol in LAM cells, suggesting that anti-estrogen therapy may be a treatment modality. Furthermore, proteases and GPNMB might be useful LAM biomarkers.

LH-Induced Steroidogenesis in the Mouse Ovary, but Not Testis, Requires Matrix Metalloproteinase 2- and 9-Mediated Cleavage of Upregulated EGF Receptor Ligands.

Oocyte maturation and cumulus cell expansion depend on luteinizing hormone (LH)-mediated upregulation of membrane-bound epidermal growth factor (EGF)-like ligands, including amphiregulin, epiregulin, and betacellulin. These ligands then transactivate the EGF receptor (EGFR) after release by matrix metalloproteinases (MMPs). However, direct measurement of released EGF-like ligands or MMPs from granulosa cells has not been formally evaluated, nor has direct identification of responsible MMPs. Here we address these issues by analyzing LH-induced steroidogenesis, which is also MMP and EGFR dependent, in freshly isolated mouse primary granulosa cells. We demonstrate a correlation between amphiregulin and epiregulin mRNA induction and steroid production in LH-treated granulosa cells as well as in ovaries of human chorionic gonadotropin-treated mice. In contrast, LH does not alter Mmp1, Mmp2, Mmp3, Mmp8, Mmp9, or Adam17 mRNA expression. We demonstrate that, in primary mouse granulosa cells, LH triggers release of soluble amphiregulin that correlates with steroid production, both of which are blocked by MMP2/9 inhibition, confirming that MMP2/9 likely regulates LH-induced amphiregulin release and downstream processes. Notably, LH does not alter secretion of MMP2/9 from primary granulosa cells, nor does it modulate MMP activity. These findings indicate that, in the ovary, LH dictates EGFR-mediated processes not by regulating MMPs, but instead by increasing EGF-like ligand availability. In contrast, LH stimulation of primary mouse Leydig cells does not induce EGF-like ligand expression or require MMP2/9 for steroidogenesis, confirming marked differences in LH receptor-induced processes in the testes. Our results suggest that MMP inhibition may be a means of attenuating excess ovarian steroid production in diseases like polycystic ovary syndrome.

Androgens regulate ovarian follicular development by increasing follicle stimulating hormone receptor and microRNA-125b expression.

Although androgen excess is considered detrimental to women's health and fertility, global and ovarian granulosa cell-specific androgen-receptor (AR) knockout mouse models have been used to show that androgen actions through ARs are actually necessary for normal ovarian function and female fertility. Here we describe two AR-mediated pathways in granulosa cells that regulate ovarian follicular development and therefore female fertility. First, we show that androgens attenuate follicular atresia through nuclear and extranuclear signaling pathways by enhancing expression of the microRNA (miR) miR-125b, which in turn suppresses proapoptotic protein expression. Second, we demonstrate that, independent of transcription, androgens enhance follicle-stimulating hormone (FSH) receptor expression, which then augments FSH-mediated follicle growth and development. Interestingly, we find that the scaffold molecule paxillin regulates both processes, making it a critical regulator of AR actions in the ovary. Finally, we report that low doses of exogenous androgens enhance gonadotropin-induced ovulation in mice, further demonstrating the critical role that androgens play in follicular development and fertility. These data may explain reported positive effects of androgens on ovulation rates in women with diminished ovarian reserve. Furthermore, this study demonstrates mechanisms that might contribute to the unregulated follicle growth seen in diseases of excess androgens such as polycystic ovary syndrome.

Uterine-specific loss of Tsc2 leads to myometrial tumors in both the uterus and lungs.

Lymphangioleiomyomatosis (LAM) is a rare disease characterized by proliferation of abnormal smooth-muscle cells in the lungs, leading to functional loss and sometimes lung transplantation. Although the origin of LAM cells is unknown, several features of LAM provide clues. First, LAM cells contain inactivating mutations in genes encoding Tsc1 or Tsc2, proteins that limit mTORC1 activity. Second, LAM tumors recur after lung transplantation, suggesting a metastatic pathogenesis. Third, LAM is found almost exclusively in women. Finally, LAM shares features with uterine leiomyomas, benign tumors of myometrial cells. From these observations, we proposed that LAM cells might originate from uterine leiomyomas containing Tsc mutations. To test our hypothesis, and to develop mouse models for leiomyoma and LAM, we targeted Tsc2 deletion primarily in uterine cells. In fact, nearly 100% of uteri from uterine-specific Tsc2 knockout mice developed myometrial proliferation and uterine leiomyomas by 12 and 24 weeks, respectively. Myometrial proliferation and mTORC1/S6 activity were abrogated by the mTORC1 inhibitor rapamycin or by elimination of sex steroid production through ovariectomy or aromatase inhibition. In ovariectomized Tsc2 null mice, mTORC1/S6 activity and myometrial growth were restored by estrogen but not progesterone. Thus, even without Tsc2, estrogen appears to be required for myometrial mTORC1/S6 signaling and proliferation. Finally, we found Tsc2 null myometrial tumors in lungs of older Tsc2 uterine-specific knockout females, suggesting that lung LAM-like myometrial lesions may indeed originate from the uterus. This mouse model may improve our understanding of LAM and leiomyomas and might lead to novel therapeutic strategies for both diseases.

Membrane receptor cross talk in steroidogenesis: recent insights and clinical implications.

Steroid production by all three major steroidogenic tissues, the adrenals, testes, and ovaries, is critical for survival and reproduction of all animals. As such, the pathways that regulate steroidogenesis are conserved between these tissues, from the steroidogenic enzymes and cofactors that synthesize steroids, to the intracellular signaling molecules and Gαs-coupled receptors that mediate the activity of these enzymes. Recent work has revealed another important conserved pathway in steroidogenesis: crosstalk between membrane G protein-coupled receptors and membrane receptor tyrosine kinases. Luteinizing hormone (LH) or adrencorticotropic hormone (ACTH) binding to their cognate Gαs-coupled membrane receptors in the gonads and adrenals, respectively, leads to cAMP-induced trans-activation of the epidermal growth factor (EGF) receptor, followed by activation of Akt and Erk signaling. These kinase signals then activate Steroidogenic Acute Regulatory (StAR) protein, which promotes steroid production. Inhibition of this pathway abrogates both LH- and ACTH-induced steroidogenesis. Interestingly, LH-induced transactivation of the EGF receptor in the ovary uniquely requires matrix metalloproteinase-mediated release of EGF receptor ligands, and inhibition of these proteases blocks LH-induced steroidogenesis. Given this unique need for matrix metalloproteinases in ovarian steroidogenesis, MMP inhibition may prove to be useful when treating diseases of excess ovarian steroid production, such as polycystic ovary syndrome.