An integrated approach to understand fluid shear stress-driven and reactive oxygen species-mediated metastasis of colon adenocarcinoma through mRNA-miRNA-lncRNA-circRNA networks.

Development of colon adenocarcinoma (COAD) metastasis involves several mediators including fluid shear stress (FSS), intracellular ROS levels, and non-coding RNAs. In our present study, we identified and investigated the role of regulatory non-coding RNA molecules specifically involved in COAD metastasis and their association with FSS and ROS. Interactions between the mRNAs associated with FSS and ROS, the corresponding microRNAs (miRNAs), long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) in COAD metastasis were used to generate the mRNA-miRNA-lncRNA-circRNA network. Experimental validation of the identified RNA hubs using quantitative real-time PCR demonstrated a direct effect of the FSS on their expression levels in cancer cells. FSS resulted in the downregulation of HMGA1 and RAN, as well as the upregulation of HSP90AA1, PMAIP1 and BIRC5. Application of shear stress also led to downregulation of hsa-miR-26b-5p and hsa-miR-34a-5p levels in HCT116 cells. Further, functional enrichment and survival analysis of the significant miRNAs, as well as the OncoPrint and the survival analyses of the selected mRNAs were performed. Subsequently, their functional role was also corroborated with existing literature. Ten significant miRNA hubs were identified, out of which hsa-miR-17-5p and hsa-miR-20a-5p were found to interact with lncRNA (CCAT2) while hsa-miR-335 was found to interact with four circRNAs. Fifteen significant miRNAs were identified in 10 different modules suggesting their importance in FSS and ROS-mediated COAD metastasis. Finally, 10 miRNAs and 3 mRNAs associated with FSS and/or ROS were identified as significant overall survival markers; 33 mRNAs were also identified as metastasis-free survival markers whereas 15 mRNAs showed > 10% gene alterations in TCGA-COAD data and may serve as promising therapeutic biomarkers in the COAD metastasis.

Influence mechanism of structure on shear mechanical deformation characteristics of loess-steel interface.

The mechanical properties of loess-steel interface are of great significance for understanding the residual strength and deformation of loess. However, the undisturbed loess has significant structural properties, while the remolded loess has weak structural properties. There are few reports on the mechanical properties of loess-steel interface from the structural point of view. This paper focused on the ring shear test between undisturbed loess as well as its remolded loess and steel interface under the same physical mechanics and test conditions (water content, shear rate and vertical pressure), and explored the influence mechanism of structure on the mechanical deformation characteristics of steel-loess interface. The results show that the shear rate has little effect on the residual strength of the undisturbed and remolded loess-steel interface. However, the water content has a significant influence on the residual strength of the loess-steel interface, moreover, the residual internal friction angle is the dominant factor supporting the residual strength of the loess-steel interface. In general, the residual strength of the undisturbed loess-steel interface is greater than that of the remolded loess specimen (for example, the maximum percentage of residual strength difference between undisturbed and remolded loess specimens under the same moisture content is 6.8%), which is because that compared with the mosaic arrangement structure of the remolded loess, the overhead arrangement structure of the undisturbed loess skeleton particles makes the loess particles on the loess-steel interface re-adjust the arrangement direction earlier and reach a stable speed relatively faster. The loess particles with angular angles in the undisturbed loess make the residual internal friction between the particles greater than the smoother particles of the remolded loess (for example, the maximum percentage of residual cohesion difference between undisturbed and remolded loess specimens under the same vertical pressure is 4.29%), and the intact cement between undisturbed loess particles brings stronger cohesion than the remolded loess particles with destroyed cement (for example, the maximum difference percentage of residual cohesion between undisturbed and remolded soil specimens under the same vertical pressure is 33.80%). The test results provide experimental basis for further revealing the influence mechanism of structure, and parameter basis for similar engineering construction.

Fluid shear stress regulates placental growth factor expression via heme oxygenase 1 and iron.

Increased fluid shear stress (FSS) is a key initiating stimulus for arteriogenesis, the outward remodeling of collateral arterioles in response to upstream occlusion. Placental growth factor (PLGF) is an important arteriogenic mediator. We previously showed that elevated FSS increases PLGF in a reactive oxygen species (ROS)-dependent fashion both in vitro and ex vivo. Heme oxygenase 1 (HO-1) is a cytoprotective enzyme that is upregulated by stress and has arteriogenic effects. In the current study, we used isolated murine mesentery arterioles and co-cultures of human coronary artery endothelial cells (EC) and smooth muscle cells (SMC) to test the hypothesis that HO-1 mediates the effects of FSS on PLGF. HO-1 mRNA was increased by conditions of increased flow and shear stress in both co-cultures and vessels. Both inhibition of HO-1 with zinc protoporphyrin and HO-1 knockdown abolished the effect of FSS on PLGF. Conversely, induction of HO-1 activity increased PLGF. To determine which HO-1 product upregulates PLGF, co-cultures were treated with a CO donor (CORM-A1), biliverdin, ferric ammonium citrate (FAC), or iron-nitrilotriacetic acid (iron-NTA). Of these FAC and iron-NTA induced an increase PLGF expression. This study demonstrates that FSS acts through iron to induce pro-arteriogenic PLGF, suggesting iron supplementation as a novel potential treatment for revascularization.

Dynamic flow priming programs allow tuning up the cell layers properties for engineered vascular graft.

Tissue engineered vascular grafts (TEVG) are potentially clear from ethical and epidemiological concerns sources for reconstructive surgery for small diameter blood vessels replacement. Here, we proposed a novel method to create three-layered TEVG on biocompatible glass fiber scaffolds starting from flat sheet state into tubular shape and to train the resulting tissue by our developed bioreactor system. Constructed tubular tissues were matured and trained under 3 types of individual flow programs, and their mechanical and biological properties were analyzed. Training in the bioreactor significantly increased the tissue burst pressure resistance (up to 18 kPa) comparing to untrained tissue. Fluorescent imaging and histological examination of trained vascular tissue revealed that each cell layer has its own individual response to training flow rates. Histological analysis suggested reverse relationship between tissue thickness and shear stress, and the thickness variation profiles were individual between all three types of cell layers. Concluding: a three-layered tissue structure similar to physiological can be assembled by seeding different cell types in succession; the following training of the formed tissue with increasing flow in a bioreactor is effective for promoting cell survival, improving pressure resistance, and cell layer formation of desired properties.

Non-coding RNA suppresses FUS aggregation caused by mechanistic shear stress on pipetting in a sequence-dependent manner.

Fused in sarcoma/translocated in liposarcoma (FUS/TLS) is a multitasking RNA/DNA binding protein. FUS aggregation is implicated in various neurodegenerative diseases. RNA was suggested to modulate phase transition of FUS. Here, we found that FUS transforms into the amorphous aggregation state as an instant response to the shear stress caused by usual pipetting even at a low FUS concentration, 100 nM. It was revealed that non-coding RNA can suppress the transformation of FUS into aggregates. The suppressive effect of RNA on FUS aggregation is sequence-dependent. These results suggested that the non-coding RNA could be a prospective suppressor of FUS aggregation caused by mechanistic stress in cells. Our finding might pave the way for more research on the role of RNAs as aggregation inhibitors, which could facilitate the development of therapies for neurodegenerative diseases.

Vulnerability to shear stress caused by altered peri-endothelial matrix is a key feature of Moyamoya disease.

Moyamoya disease (MMD) is characterized by progressive bilateral stenotic changes in the terminal portion of the internal carotid arteries. Although RNF213 was identified as a susceptibility gene for MMD, the exact pathogenesis remains unknown. Immunohistochemical analysis of autopsy specimens from a patient with MMD revealed marked accumulation of hyaluronan and chondroitin sulfate (CS) in the thickened intima of occlusive lesions of MMD. Hyaluronan synthase 2 was strongly expressed in endothelial progenitor cells in the thickened intima. Furthermore, MMD lesions showed minimal staining for CS and hyaluronan in the endothelium, in contrast to control endothelium showing positive staining for both. Glycosaminoglycans of endothelial cells derived from MMD and control induced pluripotent stem cells demonstrated a decreased amount of CS, especially sulfated CS, in MMD. A computational fluid dynamics model showed highest wall shear stress values in the terminal portion of the internal carotid artery, which is the predisposing region in MMD. Because the peri-endothelial extracellular matrix plays an important role in protection, cell adhesion and migration, an altered peri-endothelial matrix in MMD may contribute to endothelial vulnerability to wall shear stress. Invading endothelial progenitor cells repairing endothelial injury would produce excessive hyaluronan and CS in the intima, and cause vascular stenosis.

High Wall Shear Stress Is Related to Atherosclerotic Plaque Rupture in the Aortic Arch of Patients with Cardiovascular Disease: A Study with Computational Fluid Dynamics Model and Non-Obstructive General Angioscopy.

AIMS: Wall shear stress (WSS) has been considered a major determinant of aortic atherosclerosis. Recently, non-obstructive general angioscopy (NOGA) was developed to visualize various atherosclerotic pathologies, including in vivo ruptured plaque (RP) in the aorta. However, the relationship between aortic RP and WSS distribution within the aortic wall is unclear. This study aimed to investigate the relationship between aortic NOGA-derived RP and the stereographic distribution of WSS by computational fluid dynamics (CFD) modeling using three-dimensional computed tomography (3D-CT) angiography. METHODS: We investigated 45 consecutive patients who underwent 3D-CT before coronary angiography and NOGA during coronary angiography. WSS in the aortic arch was measured by CFD analysis based on the finite element method using uniform inlet and outlet flow conditions. Aortic RP was detected by NOGA. RESULTS: Patients with a distinct RP showed a significantly higher maximum WSS value in the aortic arch than those without aortic RP (56.2±30.6 Pa vs 36.2±19.8 Pa, p=0.017), no significant difference was noted in the mean WSS between those with and without aortic RP. In a multivariate logistic regression analysis, the presence of a maximum WSS value more than a specific value was a significant predictor of aortic RP (odds ratio 7.21, 95% confidence interval 1.78-37.1,p=0.005). CONCLUSIONS: Aortic RP detected by NOGA was strongly associated with a higher maximum WSS in the aortic arch derived by CFD using 3D-CT. The maximum WSS value may have an important role in the underlying mechanism of not only aortic atherosclerosis, but also aortic RP.

Stearoyl-CoA Desaturase-1 Attenuates the High Shear Force Damage Effect on Human MG63 Osteosarcoma Cells.

Mechanical regulation is known as an important regulator in cancer progression and malignancy. High shear force has been found to inhibit the cell cycle progression and result in cell death in various cancer cells. Stearoyl-CoA desaturase (SCD)-1, one of the important lipogenic enzymes, has recently been indicated as a potential pharmaceutical target in cancer therapy. In this study, we determined whether the cell fate control of shear force stimulation is through regulating the SCD-1 expression in cancer cells. Human MG63 osteosarcoma cells were used in this study. 2 and 20 dynes/cm2 shear forces were defined as low and high intensities, respectively. A SCD-1 upregulation in human MG63 osteosarcoma cells under 20, but not 2, dynes/cm2 shear force stimulation was shown, and this induction was regulated by Smad1/5 and peroxisome proliferator-activated receptor δ (PPARδ) signaling. Moreover, gene knockdown of PPARδ and SCD-1 in human MG63 osteosarcoma cells attenuated the differentiation inhibition and resulted in much more cell death of high shear force initiation. The present study finds a possible auto-protective role of SCD-1 upregulation in high shear force-damaged human MG63 osteosarcoma cells. However, its detailed regulation in the cancer fate decision of high shear force should be further examined.

Coronary arteries hemodynamics: effect of arterial geometry on hemodynamic parameters causing atherosclerosis.

Coronary arteries have high curvatures, and hence, flow through them causes disturbed flow patterns, resulting in stenosis and atherosclerosis. This in turn decreases the myocardial flow perfusion, causing myocardial ischemia and infarction. Therefore, in order to understand the mechanisms of these phenomena caused by high curvatures and branching of coronary arteries, we have conducted elaborate hemodynamic analysis for both (i) idealized coronary arteries with geometrical parameters representing realistic curvatures and stenosis and (ii) patient-specific coronary arteries with stenoses. Firstly, in idealized coronary arteries with approximated realistic arterial geometry representative of their curvedness and stenosis, we have computed the hemodynamic parameters of pressure drop, wall shear stress (WSS) and wall pressure gradient (WPG), and their association with the geometrical parameters of curvedness and stenosis. Secondly, we have similarly determined the wall shear stress and wall pressure gradient distributions in four patient-specific curved stenotic right coronary arteries (RCAs), which were reconstructed from medical images of patients diagnosed with atherosclerosis and stenosis; our results show high WSS and WPG regions at the stenoses and inner wall of the arterial curves. This paper provides useful insights into the causative mechanisms of the high incidence of atherosclerosis in coronary arteries. It also provides guidelines for how simulation of blood flow in patient's coronary arteries and determination of the hemodynamic parameters of WSS and WPG can provide a medical assessment of the risk of development of atherosclerosis and plaque formation, leading to myocardial ischemia and infarction. The novelty of our paper is in our showing how in actual coronary arteries (based on their CT imaging) curvilinearity and narrowing complications affect the computed WSS and WPG, associated with risk of atherosclerosis. This is very important for cardiologists to be able to properly take care of their patients and provide remedial measures before coronary complications lead to myocardial infarctions and necessitate stenting or coronary bypass surgery. We want to go one step further and provide clinical application of our research work. For that, we are offering to cardiologists worldwide to carry out hemodynamic analysis of the medically imaged coronary arteries of their patients and compute the values of the hemodynamic parameters of WSS and WPG, so as to provide them an assessment of the risk of atherosclerosis for their patients. Graphical abstract Theme and aims: Coronary arteries have high curvatures, and hence flow through them causes disturbed flow patterns, resulting in stenosis and atherosclerosis. This in turn decreases the myocardial flow perfusion, causing myocardial ischemia and infarction. Therefore, in order to understand the mechanisms of these phenomena caused by high curvatures and branching of coronary arteries, we have conducted elaborate hemodynamic analysis for both (i) idealized coronary arteries with geometrical parameters representing curvatures and stenosis, and (ii) patient-specific coronary arteries with stenoses. Methods and results: Firstly, in idealized coronary arteries with approximated realistic arterial geometry representative of their curvedness and stenosis, we have computed the hemodynamic parameters of pressure drop, wall shear stress (WSS) and wall pressure gradient (WPG), and their association with the geometrical parameters of curvedness and stenosis. Then, we have determined the wall shear stress and wall pressure gradient distributions in four patient-specific curved stenotic right coronary arteries (RCAs), that were reconstructed from medical images of patients diagnosed with atherosclerosis and stenosis, as illustrated in Figure 1, in which the locations of the stenoses are highlighted by arrows. Figure 1: Three-dimensional CT visualization of arteries in patients with suspected coronary disease. The arteries can be seen as a combination of various curved segments with stenoses at unspecific locations highlighted by arrows. Our results show high WSS and WPG regions at the stenoses and inner wall of the arterial curves, as depicted in Figure 2. Therein, the encapsulations show (i) high WSS, and (ii) high WPG regions at the stenosis and inner wall of the arterial curves. Figure 2: WSS and WPG surface plot of realistic arteries (a), (b), (c) and (d), wherein the small squared parts are enlarged to show the detailed localized contour plots at the stenotic regions. Therein, the circular encapsulations show (i) high WSS and (ii) high WPG regions at the stenosis and inner wall of the arterial curves. Conclusion and novelty: This paper provides useful insights into the causative mechanisms of the high incidence of atherosclerosis in coronary arteries. It also provides guidelines for how simulation of blood flow in patient coronary arteries and determination of the hemodynamic parameters of WSS and WPG can provide a medical assessment of the risk of development of atherosclerosis and plaque formation, leading to myocardial ischemia and infarction. The novelty of our paper is our showing how in actual coronary arteries (based on their CT imaging), curvilinearity and narrowing complications affect the computed WSS and WPG associated with risk of atherosclerosis. This is very important for cardiologists to be able to properly take care of their patients and provide remedial measures before coronary complications lead to myocardial infarctions and necessitate stenting or coronary bypass surgery.

Bone ongrowth and mechanical fixation of implants in cortical and cancellous bone.

BACKGROUND: What is the right surface for an implant to achieve biological fixation? Surface technologies can play important roles in encouraging interactions between the implant surface and the host bone to achieve osseointegration. Preclinical animal models provide important insight into in vivo performance related to bone ongrowth and implant fixation. METHODS: A large animal model was used to compare the in vivo response of HA and plasma-sprayed titanium coatings in a well-reported adult ovine model to evaluate bone ongrowth in terms of mechanical properties in cortical sites, and histology and histomorphometry in cortical and cancellous sites at 4 and 12 weeks. RESULTS: Titanium plasma-sprayed surfaces outperformed the HA-coated samples in push-out testing in cortical sites while both surfaces supported new bone ongrowth and remodeling in cortical and cancellous sites. CONCLUSIONS: While both HA and Ti plasma provided an osteoconductive surface for bone ongrowth, the Ti plasma provided a more robust bone-implant interface that ideally would be required for load transfer and implant stability in the longer term.

Wall Stress Distribution in Bicuspid Aortic Valve-Associated Ascending Thoracic Aortic Aneurysms.

BACKGROUND: Bicuspid aortic valve-associated ascending thoracic aortic aneurysms (BAV-aTAAs) carry a risk of acute type A dissection. Biomechanically, dissection may occur when wall stress exceeds wall strength. Our aim was to develop patient-specific computational models of BAV-aTAAs to determine magnitudes of wall stress by anatomic regions. METHODS: Patients with BAV-aTAA diameter greater than 4.5 cm (n = 41) underwent electrocardiogram-gated computed tomography angiography. Three-dimensional aneurysm geometries were reconstructed after accounting for prestress and loaded to systemic pressure. Finite element analyses were performed with fiber-embedded hyperelastic material model using LS-DYNA software (LSTC Inc, Livermore, CA) to obtain wall stress distributions. The 99th percentile longitudinal and circumferential stresses were determined at systole. RESULTS: The 99th percentile longitudinal wall stresses for BAV-aTAAs at sinuses of Valsalva, sinotubular junction (STJ), and ascending aorta were 361 ± 59.8 kPa, 295 ± 67.2 kPa, and 224 ± 37.6 kPa, respectively, with significant differences in ascending aorta vs sinuses (P< 1 × 10-13) and STJ (P < 1 × 10-6). The 99th percentile circumferential wall stresses were 474 ± 88.2 kPa, 634 ± 181.9 kPa, and 381 ± 54.0 kPa for sinuses, the STJ, and the ascending aorta, respectively, with significant differences in the ascending aorta vs sinuses (P = .002) and STJ (P < 1 × 10-13). CONCLUSIONS: Wall stresses, both circumferential and longitudinal, were greater in the aortic root, sinuses, and STJ than in the ascending aorta on BAV-aTAAs. These results fill a fundamental knowledge gap regarding biomechanical stress distribution in BAV-aTAA patients, which when related to wall strength may provide prognostication of aTAA dissection risk by patient-specific modeling.

High shear stress amplitude in combination with prolonged stimulus duration determine induction of osteoclast formation by hematopoietic progenitor cells.

To date, it is unclear how fluid dynamics stimulate mechanosensory cells to induce an osteoprotective or osteodestructive response. We investigated how murine hematopoietic progenitor cells respond to 2 minutes of dynamic fluid flow stimulation with a precisely controlled sequence of fluid shear stresses. The response was quantified by measuring extracellular adenosine triphosphate (ATP), immunocytochemistry of Piezo1, and sarcoplasmic/endoplasmic Ca2+ reticulum ATPase 2 (SERCA2), and by the ability of soluble factors produced by mechanically stimulated cells to modulate osteoclast differentiation. We rejected our initial hypothesis that peak wall shear stress rate determines the response of hematopoietic progenitor cells to dynamic fluid shear stress, as it had only a minor correlation with the abovementioned parameters. Low stimulus amplitudes corresponded to activation of Piezo1, SERCA2, low concentrations of extracellular ATP, and inhibition of osteoclastogenesis and resorption area, while high amplitudes generally corresponded to osteodestructive responses. At a given amplitude (3 Pa) and waveform (square), the duration of individual stimuli (duty cycle) showed a strong correlation with the release of ATP and osteoclast number and resorption area. Collectively, our data suggest that hematopoietic progenitor cells respond in a viscoelastic manner to loading, since a combination of high shear stress amplitude and prolonged duty cycle is needed to trigger an osteodestructive response. PLAIN LANGUAGE SUMMARY: In case of painful joints or missing teeth, the current intervention is to replace them with an implant to keep a high-quality lifestyle. When exercising or chewing, the cells in the bone around the implant experience mechanical loading. This loading generally supports bone formation to strengthen the bone and prevent breaking, but can also stimulate bone loss when the mechanical loading becomes too high around orthopedic and dental implants. We still do not fully understand how cells in the bone can distinguish between mechanical loading that strengthens or weakens the bone. We cultured cells derived from the bone marrow in the laboratory to test whether the bone loss response depends on (i) how fast a mechanical load is applied (rate), (ii) how intense the mechanical load is (amplitude), or (iii) how long each individual loading stimulus is applied (duration). We mimicked mechanical loading as it occurs in the body, by applying very precisely controlled flow of fluid over the cells. We found that a mechanosensitive receptor Piezo1 was activated by a low amplitude stimulus, which usually strengthens the bone. The potential inhibitor of Piezo1, namely SERCA2, was only activated by a low amplitude stimulus. This happened regardless of the rate of application. At a constant high amplitude, a longer duration of the stimulus enhanced the bone-weakening response. Based on these results we deduce that a high loading amplitude tends to be bone weakening, and the longer this high amplitude persists, the worse it is for the bone.

Patient-specific Hemodynamics of Severe Carotid Artery Stenosis Before and After Endarterectomy Examined by 4D Flow MRI.

Carotid endarterectomy (CEA) influences the carotid endoluminal anatomy, which results in hemodynamic changes before and after surgery. We investigated the hemodynamics of severe carotid artery stenosis before and after conventional endarterectomy with/without patch repair. An in vitro experiment utilizing carotid phantoms, which underwent a procedure that emulated CEA with/without the patch repair, was performed with a high-spatiotemporal resolution using 4D flow MRI. We evaluated an abnormal region of carotids, which consists of the normalized time-averaged wall shear stress (NTA|WSS|) and the oscillatory shear index (OSI), to account for continuous high-shear regions (high NTA|WSS| and low OSI) and chaotic low-shear regions, i.e., stenosis-prone regions (low NTA|WSS| and high OSI). The use of normalized hemodynamic parameters (e.g., NTA|WSS|) allowed comparison of diverse cases with different conditions of hemodynamics and vessel geometry. We observed that the stenosis-prone regions of the carotids with patches were noticeably larger than the corresponding regions in no-patch carotids. A large recirculating flow zone found in the stenosis-prone region of the internal carotid artery (ICA) of the postoperative carotids with patches partially blocks the flow path into ICA, and consequently the flow rate was not recovered after surgery unlike an expectation.

Coxsackievirus and adenovirus receptor mediates the responses of endothelial cells to fluid shear stress.

Endothelial mechanotransduction by fluid shear stress (FSS) modulates endothelial function and vascular pathophysiology through mechanosensors on the cell membrane. The coxsackievirus and adenovirus receptor (CAR) is not only a viral receptor but also a component of tight junctions and plays an important role in tissue homeostasis. Here, we demonstrate the expression, regulatory mechanism, and role of CAR in vascular endothelial cells (ECs) under FSS conditions. Disturbed flow increased, whereas unidirectional laminar shear stress (LSS) decreased, CAR expression in ECs through the Krüppel-like factor 2 (KLF2)/activator protein 1 (AP-1) axis. Deletion of CAR reduced the expression of proinflammatory genes and endothelial inflammation induced by disturbed flow via the suppression of NF-κB activation. Consistently, disturbed flow-induced atherosclerosis was reduced in EC-specific CAR KO mice. CAR was found to be involved in endothelial mechanotransduction through the regulation of platelet endothelial cell adhesion molecule 1 (PECAM-1) phosphorylation. Our results demonstrate that endothelial CAR is regulated by FSS and that this regulated CAR acts as an important modulator of endothelial mechanotransduction by FSS.

Shear and Dynamic Compression Modulates the Inflammatory Phenotype of Human Monocytes in vitro.

Monocytes and their derived macrophages are found at the site of remodeling tissue, such as fracture hematoma, that is exposed to mechanical forces and have been previously implicated in the reparative response. However, the mechanoresponsive of monocytes and macrophages to skeletal tissue-associated mechanical forces and their subsequent contribution to skeletal repair remains unclear. The aim of this study was to investigate the potential of skeletal tissue-associated loading conditions to modulate human monocyte activation and phenotype. Primary human monocytes or the human monocyte reporter cell line, THP1-Blue, were encapsulated in agarose and exposed to a combination of shear and compressive loading for 1 h a day for 3 consecutive days. Exposure of monocytes to mechanical loading conditions increased their pro-inflammatory gene and protein expression. Exposure of undifferentiated monocytes to mechanical loading conditions significantly upregulated gene expression levels of interleukin(IL)-6 and IL-8 compared to free swelling controls. Additionally, multiaxial loading of unstimulated monocytes resulted in increased protein secretion of TNF-α (17.1 ± 8.9 vs. 8 ± 7.4 pg/ml) and MIP-1α (636.8 ± 471.1 vs. 124.1 ± 40.1 pg/ml), as well as IL-13 (42.1 ± 19.8 vs. 21.7 ± 13.6) compared monocytes cultured under free-swelling conditions. This modulatory effect was observed irrespective of previous activation with the M1/pro-inflammatory differentiation stimuli lipopolysaccharide and interferon-γ or the M2/anti-inflammatory differentiation factor interleukin-4. Furthermore, mechanical shear and compression were found to differentially regulate nitric oxide synthase 2 (NOS2) and IL-12B gene expression as well as inflammatory protein production by THP1-Blue monocytes. The findings of this study indicate that human monocytes are responsive to mechanical stimuli, with a modulatory effect of shear and compressive loading observed toward pro-inflammatory mediator production. This may play a role in healing pathways that are mechanically regulated. An in depth understanding of the impact of skeletal tissue-associated mechanical loading on monocyte behavior may identify novel targets to maximize inflammation-mediated repair mechanisms.

Monitoring of Autophagy and Cell Volume Regulation in Kidney Epithelial Cells in Response to Fluid Shear Stress.

Fluidic shear stress applied to epithelial cells inside the kidney tubules affects cell size in an autophagy-related manner. Here, we describe the technical equipment that we routinely use to apply shear stress on cells, as well as immunoblotting, immunofluorescence, and three-dimensional cell volume reconstruction techniques used in analysis of the influence of this stress on cells and cellular components. By pointing out details of experimental techniques and potential pitfalls, this review will serve as a guide for those interested in study of how shear stress influences cells.

Shear-Induced Amyloid Formation in the Brain: IV. Effects on Synapses Surrounding Senile Plaque and in Plaque-Free Regions.

Amyloid-ß oligomers (AßO) have been proposed as neurotoxins in the synaptic dysfunction that precedes Alzheimer's disease symptoms. Human and animal model studies report that senile plaques contain a halo of AßO molecules surrounding these plaques. A far smaller number of oligomers are distributed widely in plaque-free regions. It has been suggested that oligomers migrate from halos to nearby synapses and are incorporated into both pre- and postsynaptic terminals. These two types of oligomers have two different toxicities when extracted and injected in animal models. This paper proposes a shear-energy based explanation for the data in these studies. Shear hypotheses in the preceding three papers in this series are applied to suggest how the hydrodynamics and resulting shear patterns explain the spatial distribution of both AßO types, the apparent synapse loss in the vicinity of plaque particles, and possible reasons for the differing toxicities. A shear-based mechanism is proposed for the preferential migration of locally shear-excited Aß molecules into the synaptic cleft. It is proposed that high energy laminar shear generated by the forced diversion of interstitial fluid around the flow-impeding plaque particle is responsible for the formation of AßOs around the plaque. It is suggested that in plaque-free regions, a different type of AßO with different toxicity is generated by lower energy shear flow around synapses, depositing AßO within the synapse from either the neuron membrane surface or by prion-like seeding within the synaptic cleft by locally-sheared Aß molecules near the synapse entry.

Fluid Shear Stress Promotes Autophagy in Hepatocellular Carcinoma Cells.

The autophagy in cancer cells is recognized as an essential hallmark of tumors, which can enhance cancer cell migration and invasion, and result in high incidence of tumor metastasis. The fluid shear stress (FSS) in tumor mechanical microenvironment plays a pivotal role in mediating the behaviors and functions of cells. In this study, the hepatocellular carcinoma cells were exposed to 1.4 dyn/cm2 FSS to explore whether FSS could induce autophagy. The results of TEM, Ad-mCherry-GFP labeled LC3B, and mRNA and protein expression of autophagy markers confirmed that FSS could induce autophagy in a time-dependent manner. Additionally, the inhibition of autophagy significantly downregulated the expression of PI3K, FAK and Rho GTPases, and attenuated the ability of cell migration, suggesting that FSS-induced autophagy depended on PI3K- FAK-Rho GTPases pathway. This study elucidated the role of FSS in inducing autophagy during tumor progression, which has emerged as a promising clinical strategy for cancer.

Preliminary biomechanical results of a novel pin configuration for external fixation of vertical shear pelvic fractures.

BACKGROUND: Vertical shear fractures are unstable and potentially life-threatening injuries that require urgent reduction and stabilization. The aim of this study was to compare the biomechanical efficacy of three different external fixation pin configurations for vertical shear pelvic fractures in a cadaveric model. We hypothesized that a modified external fixation pin configuration with a crestal (CR) pin in the stable hemipelvis and bilateral supra-acetabular (SA) pins provides the greatest overall stability to axial loading. METHODS: The force to failure within a standard standing axial load (maximum 650 N) was tested on 10 human cadaveric pelvises with vertical shear fractures. Three pin configurations were compared including iliac crest (IC), SA and a modified SA frame with a third CR pin on the stable hemipelvis. Both displacement at the posterior pelvis at 650 N and force to failure of >25 mm displacement was recorded. RESULTS: The mean force to failure was highest with CR (499 N), then IC (350 N) and then SA (265 N) pin configurations, being statistically non-significant (P = 0.165). The minimum force to failure followed a similar trend with 296, 68 and 43 N for CR, IC and SA, respectively. About 1/4 CR, 1/4 IC and 2/9 SA pins sustained 650 N or more without failure. CONCLUSION: It was shown that this new design may reliably withstand a seated physiological load of 250 N. However, none of the three pin configurations tested can reliably withstand a standing load of 650 N. Further experiments are needed to quantify these findings under physiological loading.

Quantifying the mechanical and histological properties of thrombus analog made from human blood for the creation of synthetic thrombus for thrombectomy device testing.

BACKGROUND: Untreated ischemic stroke can lead to severe morbidity and death, and as such, there are numerous endovascular blood-clot removal (thrombectomy) devices approved for human use. Human thrombi types are highly variable and are typically classified in qualitative terms - 'soft/red,' 'hard/white,' or 'aged/calcified.' Quantifying human thrombus properties can accelerate the development of thrombus analogs for the study of thrombectomy outcomes, which are often inconsistent among treated patients. METHODS: 'Soft'human thrombi were created from blood samples ex vivo (ie, human blood clotted in sample vials) and tested for mechanical properties using a hybrid rheometer material testing system. Synthetic thrombus materials were also mechanically tested and compared with the 'soft' human blood clots. RESULTS: Mechanical testing quantified the shear modulus and dynamic (elastic) modulus of volunteer human thrombus samples. This data was used to formulate a synthetic blood clot made from a composite polymer hydrogel of polyacrylamide and alginate (PAAM-Alg). The PAAM-Alg interpenetrating network of covalently and ionically cross-linked polymers had tunable elastic and shear moduli properties and shape memory characteristics. CONCLUSIONS: Due to its adjustable properties, PAAM-Alg can be modified to mimic various thrombi classifications. Future studies will include obtaining and quantitatively classifying patient thrombectomy samples and altering the PAAM-Alg to mimic the results for use with in vitro thrombectomy studies.