ALBERT

Description: Vascular endothelium plays an important role in regulating the transendothelial migration of polymorphonuclear leukocytes (PMNs). In this study, the intracellular calcium ion ([Ca2+]i) signaling of endothelial cells (ECs) during PMN transmigration was examined at the single-cell level. Human umbilical vein ECs were cultured on a thin layer of collagen gel. The ECs were labeled with fura-2, immersed in formyl-Met-Leu-Phe, and subsequently perfused with fresh buffer to establish a gradient of chemoattractant across the EC monolayer. The entire process of PMN rolling on, adhering to, and transmigrating across the EC monolayer was recorded under both phase-contrast and fluorescence optics. The data showed the following: (1) At high concentration (approximately 3 × 106/mL), both PMN suspension and its supernatant stimulated frequent EC [Ca2+]i elevations across the monolayer; (2) when used at lower concentration (approximately 5 × 105/mL) to avoid the interference of soluble factors, PMN transmigration, but not rolling or adhesion, was accompanied by EC [Ca2+]i elevation; (3) the latter EC [Ca2+]i elevation occurred simultaneously in ECs adjacent to the transmigration site, but not in those that were not in direct contact with the transmigrating PMNs; (4) this EC [Ca2+]i elevation was an initial and required event for PMN transmigration; and (5) PMNs pretreated with 5,5′-dimethyl-1,2-bis(2-aminophenoxy)ethane-N, N, N′, N′-tetraacetic acid transmigrated with the accompanying EC [Ca2+]i elevation, but they became elongated in the collagen gel. In conclusion, PMNs induce adjacent EC [Ca2+]i signaling, which apparently mediates the “gating” step for their subsequent transmigration.

Description: This study was conducted to elucidate whether microRNA-29a (miR-29a) and/or together with transplantation of mesenchymal stem cells isolated from umbilical cord Wharton’s jelly (uMSCs) could aid in skeletal muscle healing and putative molecular mechanisms. We established a skeletal muscle ischemic injury model by injection of a myotoxin bupivacaine (BPVC) into gastrocnemius muscle of C57BL/6 mice. Throughout the angiogenic and fibrotic phases of muscle healing, miR-29a was considerably downregulated in BPVC-injured gastrocnemius muscle. Overexpressed miR-29a efficaciously promoted human umbilical vein endothelial cells proliferation and capillary-like tube formation in vitro, crucial steps for neoangiogenesis, whereas knockdown of miR-29a notably suppressed those endothelial functions. Remarkably, overexpressed miR-29a profitably elicited limbic flow perfusion and estimated by Laser Dopple. MicroRNA-29a motivated perfusion recovery through abolishing the tissue inhibitor of metalloproteinase (TIMP)-2, led great numbers of pro-angiogenic matrix metalloproteinases (MMPs) to be liberated from bondage of TIMP, thus reinforced vascular development. Furthermore, engrafted uMSCs also illustrated comparable effect to restore the flow perfusion and augmented vascular endothelial growth factors-A, -B, and -C expression. Notably, the combination of miR29a and the uMSCs treatments revealed the utmost renovation of limbic flow perfusion. Amplified miR-29a also adequately diminished the collagen deposition and suppressed broad-wide miR-29a targeted extracellular matrix components expression. Consistently, miR-29a administration intensified the relevance of uMSCs to abridge BPVC-aggravated fibrosis. Our data support that miR-29a is a promising pro-angiogenic and anti-fibrotic microRNA which delivers numerous advantages to endorse angiogenesis, perfusion recovery, and protect against fibrosis post injury. Amalgamation of nucleic acid-based strategy (miR-29a) together with the stem cell-based strategy (uMSCs) may be an innovative and eminent strategy to accelerate the healing process post skeletal muscle injury.

Description: Skeletal muscle injury presents a challenging traumatological dilemma, and current therapeutic options remain mediocre. This study was designed to delineate if engraftment of mesenchymal stem cells derived from umbilical cord Wharton’s jelly (uMSCs) could aid in skeletal muscle healing and persuasive molecular mechanisms. We established a skeletal muscle injury model by injection of myotoxin bupivacaine (BPVC) into quadriceps muscles of C57BL/6 mice. Post BPVC injection, neutrophils, the first host defensive line, rapidly invaded injured muscle and induced acute inflammation. Engrafted uMSCs effectively abolished neutrophil infiltration and activation, and diminished neutrophil chemotaxis, including Complement component 5a (C5a), Keratinocyte chemoattractant (KC), Macrophage inflammatory protein (MIP)-2, LPS-induced CXC chemokine (LIX), Fractalkine, Leukotriene B4 (LTB4), and Interferon-γ, as determined using a Quantibody Mouse Cytokine Array assay. Subsequently, uMSCs noticeably prevented BPVC-accelerated collagen deposition and fibrosis, measured by Masson’s trichrome staining. Remarkably, uMSCs attenuated BPVC-induced Transforming growth factor (TGF)-β1 expression, a master regulator of fibrosis. Engrafted uMSCs attenuated TGF-β1 transmitting through interrupting the canonical Sma- And Mad-Related Protein (Smad)2/3 dependent pathway and noncanonical Smad-independent Transforming growth factor beta-activated kinase (TAK)-1/p38 mitogen-activated protein kinases signaling. The uMSCs abrogated TGF-β1-induced fibrosis by reducing extracellular matrix components including fibronectin-1, collagen (COL) 1A1, and COL10A1. Most importantly, uMSCs modestly extricated BPVC-impaired gait functions, determined using CatWalk™ XT gait analysis. This work provides several innovative insights into and molecular bases for employing uMSCs to execute therapeutic potential through the elimination of neutrophil-mediated acute inflammation toward protecting against fibrosis, thereby rescuing functional impairments post injury.

Description: Vascular endothelium plays an important role in regulating the transendothelial migration of polymorphonuclear leukocytes (PMNs). In this study, the intracellular calcium ion ([Ca2+]i) signaling of endothelial cells (ECs) during PMN transmigration was examined at the single-cell level. Human umbilical vein ECs were cultured on a thin layer of collagen gel. The ECs were labeled with fura-2, immersed in formyl-Met-Leu-Phe, and subsequently perfused with fresh buffer to establish a gradient of chemoattractant across the EC monolayer. The entire process of PMN rolling on, adhering to, and transmigrating across the EC monolayer was recorded under both phase-contrast and fluorescence optics. The data showed the following: (1) At high concentration (approximately 3 × 106/mL), both PMN suspension and its supernatant stimulated frequent EC [Ca2+]i elevations across the monolayer; (2) when used at lower concentration (approximately 5 × 105/mL) to avoid the interference of soluble factors, PMN transmigration, but not rolling or adhesion, was accompanied by EC [Ca2+]i elevation; (3) the latter EC [Ca2+]i elevation occurred simultaneously in ECs adjacent to the transmigration site, but not in those that were not in direct contact with the transmigrating PMNs; (4) this EC [Ca2+]i elevation was an initial and required event for PMN transmigration; and (5) PMNs pretreated with 5,5′-dimethyl-1,2-bis(2-aminophenoxy)ethane-N, N, N′, N′-tetraacetic acid transmigrated with the accompanying EC [Ca2+]i elevation, but they became elongated in the collagen gel. In conclusion, PMNs induce adjacent EC [Ca2+]i signaling, which apparently mediates the “gating” step for their subsequent transmigration.

Description: Most existing evidence regarding junction protein movements during transendothelial migration of leukocytes comes from taking postfixation snap shots of the transendothelial migration process that happens on a cultured endothelial monolayer. In this study, we used junction protein–specific antibodies that did not interfere with the transendothelial migration to examine the real-time movements of vascular endothelial–cadherin (VE-cadherin) and platelet/endothelial cell adhesion molecule-1 (PECAM-1) during transmigration of polymorphonuclear leukocytes (PMNs) either through a cultured endothelial monolayer or through the endothelium of dissected human umbilical vein tissue. In either experimental model system, both junction proteins showed relative movements, not transient disappearance, at the PMN transmigration sites. VE-cadherin moved away to different ends of the transmigration site, whereas PECAM-1 opened to surround the periphery of a transmigrating PMN. Junction proteins usually moved back to their original positions when the PMN transmigration process was completed in less than 2 minutes. The relative positions of some junction proteins might rearrange to form a new interendothelial contour after PMNs had transmigrated through multicellular corners. Although transmigrated PMNs maintained good mobility, they only moved laterally underneath the vascular endothelium instead of deeply into the vascular tissue. In conclusion, our results obtained from using either cultured cells or vascular tissues showed that VE-cadherin–containing adherent junctions were relocated aside, not opened or disrupted, whereas PECAM-1–containing junctions were opened during PMN transendothelial migration.

Description: Despite the distant metastasis of cervical cancer cells being a prominent cause of mortality, neither the metastasis capacity nor the in vitro conditions mimicking adhesion of cervical cancer cells to endothelial cells have been fully elucidated. Circulating metastatic cancer cells undergo transendothelial migration and invade normal organs in distant metastasis; however, the putative molecular mechanism remains largely uncertain. In this study, we describe the use of an in vitro parallel-plate flow chamber to simulate the dynamic circulation stress on cervical cancer cells and elucidate their vascular adhesion and metastasis. We isolate the viable and shear stress-resistant (SSR) cervical cancer cells for mechanistic studies. Remarkably, the identified SSR-HeLa and SSR-CaSki exhibited high in vitro adhesive and metastatic activities. Hence, a consistently suppressed miR-128 level was revealed in SSR cell clones compared to those of parental wild-type (WT) cells. Overexpressed miR-128 attenuated SSR-HeLa cells’ adherence to human umbilical cord vein endothelial cells (HUVECs); in contrast, suppressed miR-128 efficiently augmented the static adhesion capacity in WT-HeLa and WT-CaSki cells. Hence, amplified miR-128 modestly abolished in vitro SSR-augmented HeLa and CaSki cell movement, whereas reduced miR-128 aggravated the migration speed in a time-lapse recording assay in WT groups. Consistently, the force expression of miR-128 alleviated the SSR-enhanced HeLa and CaSki cell mobility in a wound healing assay. Notably, miR-128 mediated SSR-enhanced HeLa and CaSki cells’ adhesion and metastasis through suppressed ITGA5, ITGB5, sLex, CEACAM-6, MMP9, and MMP23 transcript levels. Our data provide evidence suggesting that miR-128 is a promising microRNA that prevented endothelial cells’ adhesion and transendothelial migration to contribute to the SSR-enhanced adhesion and metastasis progression under a parallel-plate flow chamber system. This indicates that the nucleoid-based miR-128 strategy may be an attractive therapeutic strategy to eliminate tumor cells resistant to circulation shear flow, prevent vascular adhesion, and preclude subsequent transendothelial metastasis.