Arterial and Venous Thromboembolic Complications in 832 Patients with BCR-ABL-Negative Myeloproliferative Neoplasms.

Arterial (ATE) and venous (VTE) thromboembolic complications are common causes of morbidity and mortality in BCR-ABL-negative myeloproliferative neoplasms (MPNs). However, there are few studies that include all MPN subtypes and focus on both MPN-associated ATE and VTE. In our single-center retrospective study of 832 MPN patients, a total of 180 first thromboembolic events occurred during a median follow-up of 6.6 years (range: 0-37.6 years), of which 105 were VTE and 75 were ATE. The probability of a vascular event at the end of the follow-up period was 36.2%, and the incidence rate for all first ATE/VTE was 2.43% patient/year. The most frequent VTE localizations were deep vein thrombosis with or without pulmonary embolism (incidence rate: 0.59% patient/year), while strokes were the most frequent ATE with an incidence rate of 0.32% patient/year. When comparing the group of patients with ATE/VTE (n = 180) and the group without such an event (n = 652) using multivariate Cox regression analyses, patients with polycythemia vera (hazard ratio [HR]: 1.660; [95% confidence interval [CI] 1.206, 2.286]) had a significantly higher risk of a thromboembolic event than the other MPN subtypes. In contrast, patients with a CALR mutation had a significantly lower risk of thromboembolism compared with JAK2-mutated MPN patients (HR: 0.346; [95% CI: 0.172, 0.699]). In summary, a high incidence of MPN-associated VTE and ATE was observed in our retrospective study. While PV patients or generally JAK2-mutated MPN patients had a significantly increased risk of such vascular events, this risk was reduced in CALR-mutated MPN patients.

The Prospective COVID-19 Post-Immunization Serological Cohort in Munich (KoCo-Impf): Risk Factors and Determinants of Immune Response in Healthcare Workers.

Antibody studies analyze immune responses to SARS-CoV-2 vaccination and infection, which is crucial for selecting vaccination strategies. In the KoCo-Impf study, conducted between 16 June and 16 December 2021, 6088 participants aged 18 and above from Munich were recruited to monitor antibodies, particularly in healthcare workers (HCWs) at higher risk of infection. Roche Elecsys® Anti-SARS-CoV-2 assays on dried blood spots were used to detect prior infections (anti-Nucleocapsid antibodies) and to indicate combinations of vaccinations/infections (anti-Spike antibodies). The anti-Spike seroprevalence was 94.7%, whereas, for anti-Nucleocapsid, it was only 6.9%. HCW status and contact with SARS-CoV-2-positive individuals were identified as infection risk factors, while vaccination and current smoking were associated with reduced risk. Older age correlated with higher anti-Nucleocapsid antibody levels, while vaccination and current smoking decreased the response. Vaccination alone or combined with infection led to higher anti-Spike antibody levels. Increasing time since the second vaccination, advancing age, and current smoking reduced the anti-Spike response. The cumulative number of cases in Munich affected the anti-Spike response over time but had no impact on anti-Nucleocapsid antibody development/seropositivity. Due to the significantly higher infection risk faced by HCWs and the limited number of significant risk factors, it is suggested that all HCWs require protection regardless of individual traits.

Toll-Like Receptor 3 Mediates Aortic Stenosis Through a Conserved Mechanism of Calcification.

BACKGROUND: Calcific aortic valve disease (CAVD) is characterized by a phenotypic switch of valvular interstitial cells to bone-forming cells. Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors at the interface between innate immunity and tissue repair. Type I interferons (IFNs) are not only crucial for an adequate antiviral response but also implicated in bone formation. We hypothesized that the accumulation of endogenous TLR3 ligands in the valvular leaflets may promote the generation of osteoblast-like cells through enhanced type I IFN signaling. METHODS: Human valvular interstitial cells isolated from aortic valves were challenged with mechanical strain or synthetic TLR3 agonists and analyzed for bone formation, gene expression profiles, and IFN signaling pathways. Different inhibitors were used to delineate the engaged signaling pathways. Moreover, we screened a variety of potential lipids and proteoglycans known to accumulate in CAVD lesions as potential TLR3 ligands. Ligand-receptor interactions were characterized by in silico modeling and verified through immunoprecipitation experiments. Biglycan (Bgn), Tlr3, and IFN-α/ß receptor alpha chain (Ifnar1)-deficient mice and a specific zebrafish model were used to study the implication of the biglycan (BGN)-TLR3-IFN axis in both CAVD and bone formation in vivo. Two large-scale cohorts (GERA [Genetic Epidemiology Research on Adult Health and Aging], n=55 192 with 3469 aortic stenosis cases; UK Biobank, n=257 231 with 2213 aortic stenosis cases) were examined for genetic variation at genes implicated in BGN-TLR3-IFN signaling associating with CAVD in humans. RESULTS: Here, we identify TLR3 as a central molecular regulator of calcification in valvular interstitial cells and unravel BGN as a new endogenous agonist of TLR3. Posttranslational BGN maturation by xylosyltransferase 1 (XYLT1) is required for TLR3 activation. Moreover, BGN induces the transdifferentiation of valvular interstitial cells into bone-forming osteoblasts through the TLR3-dependent induction of type I IFNs. It is intriguing that Bgn-/-, Tlr3-/-, and Ifnar1-/- mice are protected against CAVD and display impaired bone formation. Meta-analysis of 2 large-scale cohorts with >300 000 individuals reveals that genetic variation at loci relevant to the XYLT1-BGN-TLR3-interferon-α/ß receptor alpha chain (IFNAR) 1 pathway is associated with CAVD in humans. CONCLUSIONS: This study identifies the BGN-TLR3-IFNAR1 axis as an evolutionarily conserved pathway governing calcification of the aortic valve and reveals a potential therapeutic target to prevent CAVD.

Bleeding complications in bcr-abl-negative myeloproliferative neoplasms (MPN): A retrospective single-center study of 829 MPN patients.

In patients with bcr-abl-negative myeloproliferative neoplasms (MPN), concerns are often raised about the use of anticoagulants because of an increased bleeding risk. However, there are few MPN studies focusing on bleeding. To investigate bleeding complications in MPN, we report our retrospective, single-center study of 829 patients with a median follow-up of 5.5 years (range: 0.1-35.6). A first bleeding event occurred in 143 of 829 patients (17.2%), corresponding to an incidence rate of 2.29% per patient/year. During the follow-up period, one out of 829 patients (0.1%) died due to bleeding. Regarding anticoagulation, most bleeding occurred in patients on antiplatelet therapies (60.1%), followed by patients on anticoagulation therapies (20.3%) and patients not on anticoagulation (19.6%). In multivariate analysis, administration of antiplatelet (HR 2.31 [1.43, 3.71]) and anticoagulation therapies (HR 4.06 [2.32, 7.09]), but not age, gender or mutation status, was associated with an increased bleeding risk. Comparing the "probability of bleeding-free survival" between the MPN subtypes, no significant difference was observed (p = 0.91, log-rank test). Our retrospective study shows that antiplatelet and anticoagulation therapies significantly increase the risk of bleeding in MPN patients without affecting mortality. However, there is no reason to refrain from guideline-conform primary or secondary anticoagulation in MPN patients.

Ruxolitinib-treated polycythemia vera patients and their risk of secondary malignancies.

Recently, there has been increased concern about a risk of secondary malignancies (SM) occurring in myelofibrosis (MF) patients receiving ruxolitinib (RUX). In polycythemia vera (PV), on the other hand, only limited data on the risk of SM under RUX treatment are available. To investigate the association between RUX therapy in PV and SM, we conducted a retrospective, single-center study that included 289 PV patients. RUX was administered to 32.9% (95/289) of patients for a median treatment duration of 48.0 months (range 1.0-101.6). Within a median follow-up of 97 months (1.0-395.0) after PV diagnosis, 24 SM occurred. Comparing the number of PV patients with RUX-associated SM (n = 10, 41.7%) with the 14 (58.3%) patients who developed SM without RUX, no significant difference (p = 0.34, chi square test) was found. No increased incidences of melanoma, lymphoma, or solid "non-skin" malignancies were observed with RUX (p = 0.31, p = 0.60, and p = 0.63, respectively, chi square test). However, significantly more NMSC occurred in association with RUX treatment (p = 0.03, chi-squared test). The "SM-free survival" was not significantly different by log rank test for all 289 patients (p = 0.65), for the patients (n = 208; 72%) receiving cytoreductive therapy (p = 0.48) or for different therapy sequences (p = 0.074). In multivariate analysis, advanced age at PV diagnosis (HR 1.062 [95% CI 1.028, 1.098]) but not administration of RUX (HR 1.068 [95% CI 0.468, 2.463]) was associated with an increased risk for SM (p = 0.005). According to this retrospective analysis, no increased risk of SM due to RUX treatment could be substantiated for PV.

Broad T Cell Targeting of Structural Proteins After SARS-CoV-2 Infection: High Throughput Assessment of T Cell Reactivity Using an Automated Interferon Gamma Release Assay.

Background: Adaptive immune responses to structural proteins of the virion play a crucial role in protection against coronavirus disease 2019 (COVID-19). We therefore studied T cell responses against multiple SARS-CoV-2 structural proteins in a large cohort using a simple, fast, and high-throughput approach. Methods: An automated interferon gamma release assay (IGRA) for the Nucleocapsid (NC)-, Membrane (M)-, Spike-C-terminus (SCT)-, and N-terminus-protein (SNT)-specific T cell responses was performed using fresh whole blood from study subjects with convalescent, confirmed COVID-19 (n = 177, more than 200 days post infection), exposed household members (n = 145), and unexposed controls (n = 85). SARS-CoV-2-specific antibodies were assessed using Elecsys® Anti-SARS-CoV-2 (Ro-N-Ig) and Anti-SARS-CoV-2-ELISA (IgG) (EI-S1-IgG). Results: 156 of 177 (88%) previously PCR confirmed cases were still positive by Ro-N-Ig more than 200 days after infection. In T cells, most frequently the M-protein was targeted by 88% seropositive, PCR confirmed cases, followed by SCT (85%), NC (82%), and SNT (73%), whereas each of these antigens was recognized by less than 14% of non-exposed control subjects. Broad targeting of these structural virion proteins was characteristic of convalescent SARS-CoV-2 infection; 68% of all seropositive individuals targeted all four tested antigens. Indeed, anti-NC antibody titer correlated loosely, but significantly with the magnitude and breadth of the SARS-CoV-2-specific T cell response. Age, sex, and body mass index were comparable between the different groups. Conclusion: SARS-CoV-2 seropositivity correlates with broad T cell reactivity of the structural virus proteins at 200 days after infection and beyond. The SARS-CoV-2-IGRA can facilitate large scale determination of SARS-CoV-2-specific T cell responses with high accuracy against multiple targets.

Photobiomodulation Response From 660 nm is Different and More Durable Than That From 980 nm.

BACKGROUND AND OBJECTIVES: Photobiomodulation (PBM) therapy uses light at various wavelengths to stimulate wound healing, grow hair, relieve pain, and more-but there is no consensus about optimal wavelengths or dosimetry. PBM therapy works through putative, wavelength-dependent mechanisms including direct stimulation of mitochondrial respiration, and/or activation of transmembrane signaling channels by changes in water activity. A common wavelength used in the visible red spectrum is ~660 nm, whereas recently ~980 nm is being explored and both have been proposed to work via different mechanisms. We aimed to gain more insight into identifying treatment parameters and the putative mechanisms involved. STUDY DESIGN/MATERIALS AND METHODS: Fluence-response curves were measured in cultured keratinocytes and fibroblasts exposed to 660 or 980 nm from LED sources. Metabolic activity was assessed using the MTT assay for reductases. ATP production, a major event triggered by PBM therapy, was assessed using a luminescence assay. To measure the role of mitochondria, we used an ELISA to measure COX-1 and SDH-A protein levels. The respective contributions of cytochrome c oxidase and ATP synthase to the PBM effects were gauged using specific inhibitors. RESULTS: Keratinocytes and fibroblasts responded differently to exposures at 660 nm (red) and 980 nm (NIR). Although 980 nm required much lower fluence for cell stimulation, the resulting increase in ATP levels was short-term, whereas 660 nm stimulation elevated ATP levels for at least 24 hours. COX-1 protein levels were increased following 660 nm treatment but were unaffected by 980 nm. In fibroblasts, SDH-A levels were affected by both wavelengths, whereas in keratinocytes only 660 nm light impacted SDH-A levels. Inhibition of ATP synthase nearly completely abolished the effects of both wavelengths on ATP synthesis. Interestingly, inhibiting cytochrome c oxidase did not prevent the rise in ATP levels in response to PBM treatment. CONCLUSION: To the best of our knowledge, this is the first demonstration of differing kinetics in response to PBM therapy at red versus NIR wavelength. We also found cell-type-specific differences in PBM therapy response to the two wavelengths studied. These findings confirm that different response pathways are involved after 660 and 980 nm exposures and suggest that 660 nm causes a more durable response. © 2021 Wiley Periodicals LLC.

Purinergic P2Y2 receptors modulate endothelial sprouting.

Purinergic P2 receptors are critical regulators of several functions within the vascular system, including platelet aggregation, vascular inflammation, and vascular tone. However, a role for ATP release and P2Y receptor signalling in angiogenesis remains poorly defined. Here, we demonstrate that blood vessel growth is controlled by P2Y2 receptors. Endothelial sprouting and vascular tube formation were significantly dependent on P2Y2 expression and inhibition of P2Y2 using a selective antagonist blocked microvascular network generation. Mechanistically, overexpression of P2Y2 in endothelial cells induced the expression of the proangiogenic molecules CXCR4, CD34, and angiopoietin-2, while expression of VEGFR-2 was decreased. Interestingly, elevated P2Y2 expression caused constitutive phosphorylation of ERK1/2 and VEGFR-2. However, stimulation of cells with the P2Y2 agonist UTP did not influence sprouting unless P2Y2 was constitutively expressed. Finally, inhibition of VEGFR-2 impaired spontaneous vascular network formation induced by P2Y2 overexpression. Our data suggest that P2Y2 receptors have an essential function in angiogenesis, and that P2Y2 receptors present a therapeutic target to regulate blood vessel growth.

Pheno-seq - linking visual features and gene expression in 3D cell culture systems.

Patient-derived 3D cell culture systems are currently advancing cancer research since they potentiate the molecular analysis of tissue-like properties and drug response under well-defined conditions. However, our understanding of the relationship between the heterogeneity of morphological phenotypes and the underlying transcriptome is still limited. To address this issue, we here introduce "pheno-seq" to directly link visual features of 3D cell culture systems with profiling their transcriptome. As prototypic applications breast and colorectal cancer (CRC) spheroids were analyzed by pheno-seq. We identified characteristic gene expression signatures of epithelial-to-mesenchymal transition that are associated with invasive growth behavior of clonal breast cancer spheroids. Furthermore, we linked long-term proliferative capacity in a patient-derived model of CRC to a lowly abundant PROX1-positive cancer stem cell subtype. We anticipate that the ability to integrate transcriptome analysis and morphological patho-phenotypes of cancer cells will provide novel insight on the molecular origins of intratumor heterogeneity.

High risk of recurrent venous thromboembolism in BCR-ABL-negative myeloproliferative neoplasms after termination of anticoagulation.

Venous thromboembolism (VTE) is a major burden in patients with BCR-ABL-negative myeloproliferative neoplasms (MPN). In addition to cytoreductive treatment anticoagulation is mandatory, but optimal duration of anticoagulation is a matter of debate. In our single center study, we retrospectively included 526 MPN patients. In total, 78 of 526 MPN patients (14.8%) had 99 MPN-associated VTE. Median age at first VTE was 52.5 years (range 23-81). During a study period of 3497 years, a VTE event rate of 1.7% per patient/year was detected. 38.4% (38/99) of all VTEs appeared before or at MPN diagnosis and 55.6% (55/99) occurred at "uncommon" sites like splanchnic or cerebral veins. MPN patients with VTEs were significantly more female (p = 0.028), JAK2 positive (p = 0.018), or had a polycythemia vera (p = 0.009). MPN patients without VTEs were more often CALR positive (p = 0.023). Total study period after first VTE was 336 years with 20 VTE recurrences accounting for a recurrence rate of 6% per patient/year. In 36 of 71 MPN patients with anticoagulation therapy after first VTE event (50.7%), prophylactic anticoagulation was terminated after a median time of 6 months (range 1-61); 13 of those 36 patients (36.1%) had a VTE recurrence after a median of 13 months (range 4-168). In contrast, only three of 35 (8.6%) patients with ongoing anticoagulation had a VTE recurrence (p = 0.0127). Thus, termination of prophylactic anticoagulation was associated with a significantly higher risk of VTE recurrence. Our data suggest that in MPN patients with VTE, a prolonged duration of anticoagulation may be beneficial.

Improvement of adipose tissue-derived cells by low-energy extracorporeal shock wave therapy.

BACKGROUND: Cell-based therapies with autologous adipose tissue-derived cells have shown great potential in several clinical studies in the last decades. The majority of these studies have been using the stromal vascular fraction (SVF), a heterogeneous mixture of fibroblasts, lymphocytes, monocytes/macrophages, endothelial cells, endothelial progenitor cells, pericytes and adipose-derived stromal/stem cells (ASC) among others. Although possible clinical applications of autologous adipose tissue-derived cells are manifold, they are limited by insufficient uniformity in cell identity and regenerative potency. METHODS: In our experimental set-up, low-energy extracorporeal shock wave therapy (ESWT) was performed on freshly obtained human adipose tissue and isolated adipose tissue SVF cells aiming to equalize and enhance stem cell properties and functionality. RESULTS: After ESWT on adipose tissue we could achieve higher cellular adenosine triphosphate (ATP) levels compared with ESWT on the isolated SVF as well as the control. ESWT on adipose tissue resulted in a significantly higher expression of single mesenchymal and vascular marker compared with untreated control. Analysis of SVF protein secretome revealed a significant enhancement in insulin-like growth factor (IGF)-1 and placental growth factor (PLGF) after ESWT on adipose tissue. DISCUSSION: Summarizing we could show that ESWT on adipose tissue enhanced the cellular ATP content and modified the expression of single mesenchymal and vascular marker, and thus potentially provides a more regenerative cell population. Because the effectiveness of autologous cell therapy is dependent on the therapeutic potency of the patient's cells, this technology might raise the number of patients eligible for autologous cell transplantation.

A DREAM Challenge to Build Prediction Models for Short-Term Discontinuation of Docetaxel in Metastatic Castration-Resistant Prostate Cancer.

PURPOSE: Docetaxel has a demonstrated survival benefit for patients with metastatic castration-resistant prostate cancer (mCRPC); however, 10% to 20% of patients discontinue docetaxel prematurely because of toxicity-induced adverse events, and the management of risk factors for toxicity remains a challenge. PATIENTS AND METHODS: The comparator arms of four phase III clinical trials in first-line mCRPC were collected, annotated, and compiled, with a total of 2,070 patients. Early discontinuation was defined as treatment stoppage within 3 months as a result of adverse treatment effects; 10% of patients discontinued treatment. We designed an open-data, crowd-sourced DREAM Challenge for developing models with which to predict early discontinuation of docetaxel treatment. Clinical features for all four trials and outcomes for three of the four trials were made publicly available, with the outcomes of the fourth trial held back for unbiased model evaluation. Challenge participants from around the world trained models and submitted their predictions. Area under the precision-recall curve was the primary metric used for performance assessment. RESULTS: In total, 34 separate teams submitted predictions. Seven models with statistically similar area under precision-recall curves (Bayes factor ≤ 3) outperformed all other models. A postchallenge analysis of risk prediction using these seven models revealed three patient subgroups: high risk, low risk, or discordant risk. Early discontinuation events were two times higher in the high-risk subgroup compared with the low-risk subgroup. Simulation studies demonstrated that use of patient discontinuation prediction models could reduce patient enrollment in clinical trials without the loss of statistical power. CONCLUSION: This work represents a successful collaboration between 34 international teams that leveraged open clinical trial data. Our results demonstrate that routinely collected clinical features can be used to identify patients with mCRPC who are likely to discontinue treatment because of adverse events and establishes a robust benchmark with implications for clinical trial design.

Inferring catalysis in biological systems.

In systems biology, one is often interested in the communication patterns between several species, such as genes, enzymes or proteins. These patterns become more recognisable when temporal experiments are performed. This temporal communication can be structured by reaction networks such as gene regulatory networks or signalling pathways. Mathematical modelling of data arising from such networks can reveal important details, thus helping to understand the studied system. In many cases, however, corresponding models still deviate from the observed data. This may be due to unknown but present catalytic reactions. From a modelling perspective, the question of whether a certain reaction is catalysed leads to a large increase of model candidates. For large networks the calibration of all possible models becomes computationally infeasible. We propose a method which determines a substantially reduced set of appropriate model candidates and identifies the catalyst of each reaction at the same time. This is incorporated in a multiple-step procedure which first extends the network by additional latent variables and subsequently identifies catalyst candidates using similarity analysis methods. Results from synthetic data examples suggest a good performance even for non-informative data with few observations. Applied on CD95 apoptotic pathway our method provides new insights into apoptosis regulation.

A novel bioreactor for the generation of highly aligned 3D skeletal muscle-like constructs through orientation of fibrin via application of static strain.

The generation of functional biomimetic skeletal muscle constructs is still one of the fundamental challenges in skeletal muscle tissue engineering. With the notion that structure strongly dictates functional capabilities, a myriad of cell types, scaffold materials and stimulation strategies have been combined. To further optimize muscle engineered constructs, we have developed a novel bioreactor system (MagneTissue) for rapid engineering of skeletal muscle-like constructs with the aim to resemble native muscle in terms of structure, gene expression profile and maturity. Myoblasts embedded in fibrin, a natural hydrogel that serves as extracellular matrix, are subjected to mechanical stimulation via magnetic force transmission. We identify static mechanical strain as a trigger for cellular alignment concomitant with the orientation of the scaffold into highly organized fibrin fibrils. This ultimately yields myotubes with a more mature phenotype in terms of sarcomeric patterning, diameter and length. On the molecular level, a faster progression of the myogenic gene expression program is evident as myogenic determination markers MyoD and Myogenin as well as the Ca(2+) dependent contractile structural marker TnnT1 are significantly upregulated when strain is applied. The major advantage of the MagneTissue bioreactor system is that the generated tension is not exclusively relying on the strain generated by the cells themselves in response to scaffold anchoring but its ability to subject the constructs to individually adjustable strain protocols. In future work, this will allow applying mechanical stimulation with different strain regimes in the maturation process of tissue engineered constructs and elucidating the role of mechanotransduction in myogenesis. STATEMENT OF SIGNIFICANCE: Mechanical stimulation of tissue engineered skeletal muscle constructs is a promising approach to increase tissue functionality. We have developed a novel bioreactor-based 3D culture system, giving the user the possibility to apply different strain regimes like static, cyclic or ramp strain to myogenic precursor cells embedded in a fibrin scaffold. Application of static mechanical strain leads to alignment of fibrin fibrils along the axis of strain and concomitantly to highly aligned myotube formation. Additionally, the pattern of myogenic gene expression follows the temporal progression observed in vivo with a more thorough induction of the myogenic program when static strain is applied. Ultimately, the strain protocol used in this study results in a higher degree of muscle maturity demonstrated by enhanced sarcomeric patterning and increased myotube diameter and length. The introduced bioreactor system enables new possibilities in muscle tissue engineering as longer cultivation periods and different strain applications will yield tissue engineered muscle-like constructs with improved characteristics in regard to functionality and biomimicry.

In vitro extracorporeal shock wave treatment enhances stemness and preserves multipotency of rat and human adipose-derived stem cells.

BACKGROUND AIMS: Adipose-derived progenitor/stem cells (ASCs) are discussed as a promising candidate for various tissue engineering approaches. However, its applicability for the clinic is still difficult due to intra- and inter-donor heterogeneity and limited life span in vitro, influencing differentiation capacity as a consequence to decreased multipotency. METHODS: Extracorporeal shock wave treatment has been proven to be a suitable clinical tool to improve regeneration of a variety of tissues for several decades, whereas the mechanisms underlying these beneficial effects remain widely unknown. RESULTS: In this study we show that human and rat adipose derived stem cells respond strongly to repetitive shock wave treatment in vitro, resulting not only in maintenance and significant elevation of mesenchymal markers (CD73, CD90, CD105), but also in significantly increased differentiation capacity towards the osteogenic and adipogenic lineage as well as toward Schwann-cell like cells even after extended time in vitro, preserving multipotency of ASCs. CONCLUSIONS: ESWT might be a promising tool to improve ASC quality for cell therapy in various tissue engineering and regenerative medicine applications.

Shock wave treatment enhances cell proliferation and improves wound healing by ATP release-coupled extracellular signal-regulated kinase (ERK) activation.

Shock wave treatment accelerates impaired wound healing in diverse clinical situations. However, the mechanisms underlying the beneficial effects of shock waves have not yet been fully revealed. Because cell proliferation is a major requirement in the wound healing cascade, we used in vitro studies and an in vivo wound healing model to study whether shock wave treatment influences proliferation by altering major extracellular factors and signaling pathways involved in cell proliferation. We identified extracellular ATP, released in an energy- and pulse number-dependent manner, as a trigger of the biological effects of shock wave treatment. Shock wave treatment induced ATP release, increased Erk1/2 and p38 MAPK activation, and enhanced proliferation in three different cell types (C3H10T1/2 murine mesenchymal progenitor cells, primary human adipose tissue-derived stem cells, and a human Jurkat T cell line) in vitro. Purinergic signaling-induced Erk1/2 activation was found to be essential for this proliferative effect, which was further confirmed by in vivo studies in a rat wound healing model where shock wave treatment induced proliferation and increased wound healing in an Erk1/2-dependent fashion. In summary, this report demonstrates that shock wave treatment triggers release of cellular ATP, which subsequently activates purinergic receptors and finally enhances proliferation in vitro and in vivo via downstream Erk1/2 signaling. In conclusion, our findings shed further light on the molecular mechanisms by which shock wave treatment exerts its beneficial effects. These findings could help to improve the clinical use of shock wave treatment for wound healing.

Parameterizing cell-to-cell regulatory heterogeneities via stochastic transcriptional profiles.

Regulated changes in gene expression underlie many biological processes, but globally profiling cell-to-cell variations in transcriptional regulation is problematic when measuring single cells. Transcriptome-wide identification of regulatory heterogeneities can be robustly achieved by randomly collecting small numbers of cells followed by statistical analysis. However, this stochastic-profiling approach blurs out the expression states of the individual cells in each pooled sample. Here, we show that the underlying distribution of single-cell regulatory states can be deconvolved from stochastic-profiling data through maximum-likelihood inference. Guided by the mechanisms of transcriptional regulation, we formulated plausible mixture models for cell-to-cell regulatory heterogeneity and maximized the resulting likelihood functions to infer model parameters. Inferences were validated both computationally and experimentally for different mixture models, which included regulatory states for multicellular function that were occupied by as few as 1 in 40 cells of the population. Importantly, when the method was extended to programs of heterogeneously coexpressed transcripts, we found that population-level inferences were much more accurate with pooled samples than with one-cell samples when the extent of sampling was limited. Our deconvolution method provides a means to quantify the heterogeneous regulation of molecular states efficiently and gain a deeper understanding of the heterogeneous execution of cell decisions.