Tendon Stem/Progenitor Cells and Their Interactions with Extracellular Matrix and Mechanical Loading.

Tendons are unique connective tissues in the sense that their biological properties are largely determined by their tendon-specific stem cells, extracellular matrix (ECM) surrounding the stem cells, mechanical loading conditions placed on the tendon, and the complex interactions among them. This review is aimed at providing an overview of recent advances in the identification and characterization of tendon stem/progenitor cells (TSPCs) and their interactions with ECM and mechanical loading. In addition, the effects of such interactions on the maintenance of tendon homeostasis and the initiation of tendon pathological conditions are discussed. Moreover, the challenges in further investigations of TSPC mechanobiology in vitro and in vivo are outlined. Finally, future research efforts are suggested, which include using specific gene knockout models and single-cell transcription profiling to enable a broad and deep understanding of the physiology and pathophysiology of tendons.

Mechanobiology of young and aging tendons: In vivo studies with treadmill running.

Tendons are unique in the sense that they are constantly subjected to large mechanical loads and that they contain tendon-specific cells, including tenocytes and tendon stem/progenitor cells. The responses of these cells to mechanical loads can be anabolic or catabolic and as a result, change the biological properties of the tendon itself that may be beneficial or detrimental. On the other hand, aging also induces aberrant changes in cellular expression of various genes and production of various types of matrix proteins in the tendon, and consequently lead to tendon degeneration and impaired healing in aging tendons; both could be improved by moderate physiological mechanical loading such as treadmill running. This article gives an overview on the mechanobiology research of young and aging animal tendons using treadmill running model. The challenges in such treadmill running studies are also discussed. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:557-565, 2018.

Hypothermia decreases cerebrospinal fluid asymmetric dimethylarginine levels in children with traumatic brain injury.

OBJECTIVES: Pathological increases in asymmetric dimethylarginine, an endogenous nitric oxide synthase inhibitor, have been implicated in endothelial dysfunction and vascular diseases. Reduced nitric oxide early after traumatic brain injury may contribute to hypoperfusion. Currently, methods to quantify asymmetric dimethylarginine in the cerebrospinal fluid have not been fully explored. We aimed to develop and validate a method to determine asymmetric dimethylarginine in the cerebrospinal fluid of a pediatric traumatic brain injury population and to use this method to assess the effects of 1) traumatic brain injury and 2) therapeutic hypothermia on this mediator. DESIGN, SETTING, AND PATIENTS: An ancillary study to a prospective, phase II randomized clinical trial of early hypothermia in a tertiary care pediatric intensive care unit for children with Traumatic brain injury admitted to Children's Hospital of Pittsburgh. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A UPLC-MS/MS method was developed and validated to quantitate asymmetric dimethylarginine. A total of 56 samples collected over 3 days with injury onset were analyzed from the cerebrospinal fluid of consented therapeutic hypothermia (n = 9) and normothermia (n = 10) children. Children undergoing diagnostic lumbar puncture (n = 5) were enrolled as controls. Asymmetric dimethylarginine was present at a quantifiable level in all samples. Mean asymmetric dimethylarginine levels were significantly increased in normothermic Traumatic brain injury children compared with that in control (0.19 ± 0.08 µmol/L and 0.11 ± 0.02 µmol/L, respectively, p = 0.01), and hypothermic children had significantly reduced mean asymmetric dimethylarginine levels (0.11 ± 0.05 µmol/L) vs. normothermic (p = 0.03) measured on day 3. Patient demographics including age, gender, and nitric oxide levels (measured as nitrite and nitrate using liquid chromatography coupled with Griess reaction) did not significantly differ between normothermia and hypothermia groups. Also, nitric oxide levels did not correlate with asymmetric dimethylarginine concentrations. CONCLUSIONS: Asymmetric dimethylarginine levels were significantly increased in the cerebrospinal fluid of traumatic brain injury children. Early hypothermia attenuated this increase. The implications of attenuated asymmetric dimethylarginine on nitric oxide synthases activity and regional cerebral blood flow after traumatic brain injury by therapeutic hypothermia deserve future study.

Cerebrospinal fluid 20-HETE is associated with delayed cerebral ischemia and poor outcomes after aneurysmal subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Delayed cerebral ischemia (DCI) is a major complication after aneurysmal subarachnoid hemorrhage (aSAH); it is manifested by changes in cerebral blood flow accompanied by neurological decline, and it results in long-term functional and neuropsychological impairment. Preclinical evidence has demonstrated that the arachidonic acid metabolite, 20-hydroxyeicosatetraenoic acid (20-HETE), affects cerebral microvascular tone and cerebral blood flow after aSAH. The purpose of this study was to determine whether cerebrospinal fluid 20-HETE levels were associated with DCI and long-term neuropsychological outcomes in aSAH patients. METHODS: Cerebrospinal fluid samples were collected twice daily through 14 days after hemorrhage on 108 acute, adult, aSAH patients. Samples were analyzed for 20-HETE via HPLC MSQ single quadrupole mass spectrometry. DCI was defined as the presence of impaired cerebral blood flow (angiographic vasospasm, elevated transcranial Dopplers, abnormal computed tomography or magnetic resonance perfusion scans) accompanied by neurological deterioration. Outcomes, including death and neuropsychological testing, were completed at 3 months after hemorrhage. RESULTS: Detectable 20-HETE levels were observed in 31% of patient samples and were associated with severity of hemorrhage (Hunt & Hess [HH], P=0.04; Fisher, P=0.05). Detection of 20-HETE was not associated with angiographic vasospasm (P=0.34); however, detectable 20-HETE was significantly associated with DCI (P=0.016). Our data also suggest that detectable 20-HETE was associated with decreased performance in 5 neuropsychological domains. CONCLUSIONS: These results provide the first clinical evidence that cerebrospinal fluid 20-HETE concentrations are associated with DCI and poor outcomes, and this provides impetus for future studies to elucidate the clinical utility of inhibiting 20-HETE formation as a novel therapeutic intervention in patients with aSAH.

Role of endothelin-1 in human aneurysmal subarachnoid hemorrhage: associations with vasospasm and delayed cerebral ischemia.

BACKGROUND: Endothelin-1 (ET-1) is a potent vasoconstrictor implicated in the pathogenesis of vasospasm and delayed cerebral ischemia (DCI) in aneurysmal subarachnoid hemorrhage (aSAH) patients. The aim of this study was to investigate the relationship between cerebrospinal fluid (CSF) ET-1 levels and angiographic vasospasm and DCI. METHODS: Patients with aSAH were consented (n = 106). Cerebral vasospasm was determined by angiography. DCI was determined by transcranial Doppler (TCD) results and/or angiogram results with corresponding clinical deterioration. CSF ET-1 levels over 14 days after the initial insult was quantified by ELISA. ET-1 analysis included a group-based trajectory analysis and ET-1 exposure rate during 24, 48, and 72 h prior to, as well as 72 h post angiography, or clinical deterioration. RESULTS: Trajectory analysis revealed two distinct groups of subjects with 56% of patients in the low ET-1 trajectory group (mean at day 1 = 0.31 pg/ml; SE = 0.04; mean at day 14 = 0.41 pg/ml; SE = 0.15) and 44% of patients in the high ET-1 trajectory group (mean at day 1 = 0.65 pg/ml; SE = 0.08; mean at day 14 = 0.61 pg/ml; SE = 0.06). Furthermore, we observed that ET-1 exposure rate 72 h before angiography and clinical spasm was a significant predictor of both angiographic vasospasm and DCI, whereas, ET-1 exposure after angiography and clinical spasm was not associated with either angiographic vasospasm or DCI. CONCLUSION: Based on these results we conclude that ET-1 concentrations are elevated in a sub-group of patients and that the acute (72 h prior to angiography and clinical neurological deterioration), but not chronic, elevations in CSF ET-1 concentrations are indicative of the pathogenic alterations of vasospasm and DCI in aSAH patients.

Mechanobiology of adult and stem cells.

Mechanical forces, including gravity, tension, compression, hydrostatic pressure, and fluid shear stress, play a vital role in human physiology and pathology. They particularly influence extracellular matrix (ECM) gene expression, ECM protein synthesis, and production of inflammatory mediators of many load-sensitive adult cells such as fibroblasts, chondrocytes, smooth muscle cells, and endothelial cells. Furthermore, the mechanical forces generated by cells themselves, known as cell traction forces (CTFs), also influence many biological processes such as wound healing, angiogenesis, and metastasis. Thus, the quantitative characterization of CTFs by qualities such as magnitude and distribution is useful for understanding physiological and pathological events at the tissue and organ levels. Recently, the effects of mechanical loads on embryonic and adult stem cells in terms of self-renewal, differentiation, and matrix protein expression have been investigated. While it seems certain that mechanical loads applied to stem cells regulate their self-renewal and induce controlled cell lineage differentiation, the detailed molecular signaling mechanisms responsible for these mechano-effects remain to be elucidated. Challenges in the fields of both adult- and stem-cell mechanobiology include devising novel experimental and theoretical methodologies to examine mechano-responses more closely to various forms of mechanical forces and mechanotransduction mechanisms of these cells in a more physiologically accurate setting. Such novel methodologies will lead to better understanding of various pathological diseases, their management, and translational applications in the ever expanding field of tissue engineering.

Mechanoregulation of gene expression in fibroblasts.

Mechanical loads placed on connective tissues alter gene expression in fibroblasts through mechanotransduction mechanisms by which cells convert mechanical signals into cellular biological events, such as gene expression of extracellular matrix components (e.g., collagen). This mechanical regulation of ECM gene expression affords maintenance of connective tissue homeostasis. However, mechanical loads can also interfere with homeostatic cellular gene expression and consequently cause the pathogenesis of connective tissue diseases such as tendinopathy and osteoarthritis. Therefore, the regulation of gene expression by mechanical loads is closely related to connective tissue physiology and pathology. This article reviews the effects of various mechanical loading conditions on gene regulation in fibroblasts and discusses several mechanotransduction mechanisms. Future research directions in mechanoregulation of gene expression are also suggested.

A new approach to study fibroblast migration.

This paper presents a new approach to study cell migration. Human tendon fibroblasts were plated on silicone membranes coated with 10 microg/ml ProNectin-F. The silicone surfaces were micro-fabricated with parallel microgrooves, with 10 microm ridge and groove width, and 3 microm groove depth. Fibroblasts grown in the microgrooves had an elongated shape and oriented along the microgroove direction. They also moved along the same direction instead of "random walk" when cells migrate on smooth culture surfaces. In response to TGF-beta1 (5 ng/ml) treatment, these fibroblasts on the microgrooved surfaces were differentiated into myofibroblasts, as judged by an elevated expression of alpha-smooth muscle actin (alpha-SMA), a specific marker for myofibroblasts. Moreover, these myofibroblasts were found to be approximately 30% less motile compared to that of untreated fibroblasts. Thus, use of microgrooved surface may be an effective approach to detect difference in cell motility because cell migration on the microgrooved surface is one dimensional and hence easier to be quantified than two-dimensional random movement on conventional smooth culture surfaces.

EP4 receptor regulates collagen type-I, MMP-1, and MMP-3 gene expression in human tendon fibroblasts in response to IL-1 beta treatment.

Tendinopathy is accompanied by inflammation, tendon matrix degradation, or both. Inflammatory cytokine IL-1beta, which is a potent inflammatory mediator, is likely present within the tendon. The purpose of this study was to determine the biological impact of IL-1beta on tendon fibroblasts by assessing the expression of cPLA(2), COX-2, PGE(2) and its receptors (EPs), collagen type-I, and MMPs. We also studied the role of the p38 MAPK pathway in IL-1beta-induced catabolic effects. We found that IL-1beta increased the expression levels of cPLA(2) and COX-2, and also increased the secretion of PGE(2). Induction of MMPs, such as MMP-1 and MMP-3 at the mRNA level, was also observed after stimulation with IL-1beta. Furthermore, the presence of IL-1beta significantly decreased the level of collagen type-I mRNA in tendon fibroblasts. These effects were found to be mediated by selective upregulation of EP(4) receptor, which is a member of G-protein-coupled receptor that transduces the PGE(2) signal. Blocking EP(4) receptor by a specific chemical inhibitor abolished IL-1beta-induced catabolic effects. These results suggest that IL-1beta-induced catabolic action on tendon fibroblasts occurs via the upregulation of two key inflammatory mediators, cPLA(2) and COX-2, which are responsible for the synthesis of PGE(2). IL-1beta further stimulates the expression of EP(4) receptor, suggesting positive feedback regulation which may lead to accelerated catabolic processes in tendon fibroblasts. Studies using pathway-specific chemical inhibitors suggest that the p38 MAPK pathway is the key signaling cascade transducing IL-1beta-mediated catabolic effects. Collectively, our findings suggest that the EP(4) receptor mediates the IL-1beta-induced catabolic metabolism via the p38 MAPK pathway in human tendon fibroblasts and may play a major role in the tendon's degenerative changes often seen in the later stages of tendinopathy.

Leukotriene B4 at low dosage negates the catabolic effect of prostaglandin E2 in human patellar tendon fibroblasts.

Tendinopathy often involves inflammation and matrix degeneration. The inflammatory mediators such as prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) are implicated in the development of tendinopathy. Therefore, the purpose of this study was to determine the effect of PGE2 and LTB4 on the proliferation of human patellar tendon fibroblasts (HPTFs), the gene expression of collagen type I, MMP-1 and MMP-3, as well as the protein secretion of these gene products by the cells. The results showed that LTB4 at low doses (0.1 and 1 nM) significantly increased cell proliferation compared to controls and LTB4 at 0.1 nM negated the PGE2-induced decrease in cell proliferation. In addition, PGE2 at 100 ng/ml significantly increased the expression of MMP-1 and MMP-3 at both mRNA and protein levels. These stimulatory effects were significantly diminished by co-treatment with LTB4 at 0.1 nM. Finally, neither PGE2 nor LTB4 treatment affected collagen type I gene expression. These results suggest that low levels of LTB4 counterbalance the negative effects mediated by PGE2 on tendon fibroblast proliferation and MMP production, which may lead to matrix degradation. Thus, our findings suggest that although LTB4 is generally thought to be pathogenic, low levels of LTB4 are actually beneficial in maintaining tendon tissue homeostasis.

SAR: flavonoids and COX-2 inhibition.

An analysis based upon structure-activity relationships (SAR) of the COX-2-inhibiting properties of flavonoids, a group of potential cancer chemopreventative agents, reveals that there is a dual structural basis for these activities. Each of these structural determinants (pharmacophores) alone is sufficient for activity. One of the pharmacophores is a 2D 6.9 A distance descriptor that spans the A and C rings and includes the 4-OXO and 7-hydroxyl moieties. The potency associated with that pharmacophore is determined by a series of structural modulators that can increase, decrease, or even abolish the COX-2-inhibiting potential associated with that pharmacophore. The second pharmacophore describes a para-substituted phenolic B ring that requires unsubstituted meta and ortho positions. Based upon this, it indicates that hydroxylation at the 4'-position and a free 5'-position are sufficient for COX-2-inhibiting activity. The potency associated with this pharmacophore is modulated by log P2 and by the molecular weight.

SAR modeling: effect of experimental ambiguity.

The applicability of SAR (structure-activity relationship) techniques to data obtained using high throughput screening (HTS) and toxicogenomic techniques is explored. The reason for this study derives from the fact that for economical and time considerations HTS bioassays may consist of single determinations, i.e. lack of duplication. This introduces an element of uncertainty. Using two different data bases of fairly complex biological phenomena (allergic contact dermatitis in humans and the induction of mutations in Salmonella), it is demonstrated that the resulting SAR models can tolerate up to 20% ambiguity in the experimental data.

Structural concepts in cancer prevention.

The notion of developing cancer preventative strategies is attractive both from a public health and from a health economic viewpoint. However, as currently visualized, this may involve dietary supplementation of publicly available foods or the ingestion of specific supplements for prolonged periods of time. In view of the fact that the outcome of such preventative strategies may as yet not be known, it is essential that the strategy is devoid of risks. Structure-activity relationship (SAR) concepts can be of use in identifying possible health hazards associated with chemoprevention. Overall, SAR can be used (1) to predict the chemopreventative potential of a chemical and to understand its mechanism of action, (2) to evaluate the toxicological liabilities of such agents and (3) to design molecules with enhanced chemopreventative potency and decreased (or abolished) toxicity. While SAR techniques currently available are appropriate to achieve these aims, the primary block to their widespread deployment is lack of sufficient experimental data of acceptable quality to perform SAR modeling. The present report analyzes the current applicability of SAR methods to cancer chemoprevention.