Pharmacological Inhibition of Cathepsin S Suppresses Abdominal Aortic Aneurysm in Mice.

OBJECTIVE: Evidence suggests that cathepsin S (CTSS), a potent mammalian elastase, participates in abdominal aortic aneurysm (AAA) formation. This study examines the hypothesis that pharmacological inhibition of CTSS with an α-ketoamide based compound 6r might suppress AAA in mice. METHODS: Experimental study of the CaCl2 induced AAA model in B6 mice and angiotensin II (AngII) infused AAA model in ApoE-/- mice. The effects of intraperitoneal administration of 6r (25 mg/kg) and vehicle every three days since one day after AAA induction were evaluated at 28 days using CaCl2 induced (n = 12 per group) and AngII infused (n = 8 per group) models. Additionally, the effects of post-treatment with 6r and vehicle from seven days or 14 days after AAA induction were evaluated at 28 days using the CaCl2 induced model (n = 6 per group). Aortic samples were harvested for histological and biochemical analyses, including cathepsin levels, Verhoeff Van Gieson staining, TUNEL assay, and immunostaining for macrophages. RESULTS: In the CaCl2 induced model, treatment with 6r suppressed aortic dilatation observed in vehicle treated controls (median: 0.58 vs. 0.92 mm; p < .001), along with reduced CTSS and cathepsin K (CTSK) levels (both p < .001), preserved elastin integrity (p < .001), fewer medial apoptotic cells (p = .012) and less macrophage infiltration (p = .041). In the AngII infused model, the aortic diameter was smaller in 6r treated mice than in vehicle treated controls (median: 0.95 vs. 1.84 mm; p = .047). The levels of CTSS (p < .001) and CTSK (p = .033) and the numbers of elastin breaks (p < .001), medial apoptotic cells (p < .001) and infiltrating macrophages (p = .030) were attenuated under 6r treatment. Finally, post-treatment with 6r from seven days (p = .046) or 14 days (p = .012) after AAA induction limited CaCl2 induced AAA. CONCLUSION: Pharmacological inhibition of CTSS by 6r suppresses AAA formation in mice. Also, post-treatment with 6r retards mouse AAA progression. These findings provide proof of concept validation for CTSS as a potential therapeutic target in AAA.

A thrombomodulin-like gene is crucial to the collective migration of epibolic blastomeres during germ layer formation and organogenesis in zebrafish.

BACKGROUND: Thrombomodulin (TM), an integral membrane protein, has long been known for its anticoagulant activity. Recent studies showed that TM displays multifaceted activities, including the involvement in cell adhesion and collective cell migration in vitro. However, whether TM contributes similarly to these biological processes in vivo remains elusive. METHODS: We adapted zebrafish, a prominent animal model for studying molecular/cellular activity, embryonic development, diseases mechanism and drug discovery, to examine how TM functions in modulating cell migration during germ layer formation, a normal and crucial physiological process involving massive cell movement in the very early stages of life. In addition, an in vivo assay was developed to examine the anti-hemostatic activity of TM in zebrafish larva. RESULTS: We found that zebrafish TM-b, a zebrafish TM-like protein, was expressed mainly in vasculatures and displayed anti-hemostatic activity. Knocking-down TM-b led to malformation of multiple organs, including vessels, heart, blood cells and neural tissues. Delayed epiboly and incoherent movement of yolk syncytial layer were also observed in early TM-b morphants. Whole mount immunostaining revealed the co-localization of TM-b with both actin and microtubules in epibolic blastomeres. Single-cell tracking revealed impeded migration of blastomeres during epiboly in TM-b-deficient embryos. CONCLUSION: Our results showed that TM-b is crucial to the collective migration of blastomeres during germ layer formation. The structural and functional compatibility and conservation between zebrafish TM-b and mammalian TM support the properness of using zebrafish as an in vivo platform for studying the biological significance and medical use of TM.

Selective ß2-AR Blockage Suppresses Colorectal Cancer Growth Through Regulation of EGFR-Akt/ERK1/2 Signaling, G1-Phase Arrest, and Apoptosis.

The stress-upregulated catecholamines-activated ß1- and ß2-adrenergic receptors (ß1/2-ARs) have been shown to accelerate the progression of cancers such as colorectal cancer (CRC). We investigated the underlying mechanism of the inhibition of ß1/2-ARs signaling for the treatment of CRC and elucidated the significance of ß2-AR expression in CRC in vitro and in clinical samples. The impacts of ß1/2-AR antagonists in CRC in vitro and CRC-xenograft in vivo were examined. We found that repression of ß2-AR but not ß1-AR signaling selectively suppressed cell viability, induced G1-phase cell cycle arrest, caused both intrinsic and extrinsic pathways-mediated apoptosis of specific CRC cells and inhibited CRC-xenograft growth in vivo. Moreover, the expression of ß2-AR was not consistent with the progression of CRC in vitro or in clinical samples. Our data evidence that the expression profiles, signaling, and blockage of ß2-AR have a unique pattern in CRC comparing to other cancers. ß2-AR antagonism selectively suppresses the growth of CRC accompanying active ß2-AR signaling, which potentially carries wild-type KRAS, in vitro and in vivo via the inhibition of ß2-AR transactivated EFGR-Akt/ERK1/2 signaling pathway. Thus, ß2-AR blockage might be a potential therapeutic strategy for combating the progressions of ß2-AR-dependent CRC.

Membrane-Bound Thrombomodulin Regulates Macrophage Inflammation in Abdominal Aortic Aneurysm.

OBJECTIVE: Thrombomodulin (TM), a glycoprotein constitutively expressed in the endothelium, is well known for its anticoagulant and anti-inflammatory properties. Paradoxically, we recently found that monocytic membrane-bound TM (ie, endogenous TM expression in monocytes) triggers lipopolysaccharide- and gram-negative bacteria-induced inflammatory responses. However, the significance of membrane-bound TM in chronic sterile vascular inflammation and the development of abdominal aortic aneurysm (AAA) remains undetermined. APPROACH AND RESULTS: Implicating a potential role for membrane-bound TM in AAA, we found that TM signals were predominantly localized to macrophages and vascular smooth muscle cells in human aneurysm specimens. Characterization of the CaCl2-induced AAA in mice revealed that during aneurysm development, TM expression was mainly localized in infiltrating macrophages and vascular smooth muscle cells. To investigate the function of membrane-bound TM in vivo, transgenic mice with myeloid- (LysMcre/TM(flox/flox)) and vascular smooth muscle cell-specific (SM22-cre(tg)/TM(flox/flox)) TM ablation and their respective wild-type controls (TM(flox/flox) and SM22-cre(tg)/TM(+/+)) were generated. In the mouse CaCl2-induced AAA model, deficiency of myeloid TM, but not vascular smooth muscle cell TM, inhibited macrophage accumulation, attenuated proinflammatory cytokine and matrix metalloproteinase-9 production, and finally mitigated elastin destruction and aortic dilatation. In vitro TM-deficient monocytes/macrophages, versus TM wild-type counterparts, exhibited attenuation of proinflammatory mediator expression, adhesion to endothelial cells, and generation of reactive oxygen species. Consistently, myeloid TM-deficient hyperlipidemic mice (ApoE(-/-)/LysMcre/TM(flox/flox)) were resistant to AAA formation induced by angiotensin II infusion, along with reduced macrophage infiltration, suppressed matrix metalloproteinase activities, and diminished oxidative stress. CONCLUSIONS: Membrane-bound TM in macrophages plays an essential role in the development of AAA by enhancing proinflammatory mediator elaboration, macrophage recruitment, and oxidative stress.

Evidence-based outcomes on diagnostic accuracy of quantitative ultrasound for assessment of pediatric osteoporosis - a systematic review.

BACKGROUND: Dual-energy absorptiometry (DXA) is the current reference standard for assessing pediatric osteoporosis; however due to its areal nature, it has limitations. Thus, quantitative ultrasound (QUS), a modality free of ionizing radiation, has been proposed as a potential surrogate for DXA. OBJECTIVE: To semi-quantitatively assess the diagnostic accuracy of QUS for evaluating pediatric osteoporosis according to the U.S. Preventive Services Task Force guidelines. MATERIALS AND METHODS: We retrieved articles on the diagnostic accuracy of quantitative US for assessing abnormal bone quality or quantity in patients of mean age ≤19 years from MEDLINE, EMBASE and Cochrane Library CCTR databases. Evidences were analyzed for reliability, construct and criterion validity, and responsiveness of quantitative US, according to the following questions: (1) How reliable is the acquisition of QUS measurements? (2) Is QUS diagnostically accurate to characterize bone strength and quality in osteoporotic children? (3) Is QUS sensitive to detect changes in bone status over time? (4) Is QUS able to predict future skeletal fractures/degeneration? Three reviewers independently evaluated the quality of reporting and methodological quality using the Standards for Reporting of Diagnostic Accuracy (STARD) and the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tools. RESULTS: Out of 262 retrieved references (215 unique), we included 28 studies (1,963 patients; 807 reported boys and 761 girls, others unspecified; reported mean age, 0-19 years). The mean quality of reporting score was "excellent" in 24/28 (86%) studies; 11/28 (39%) studies had "adequate" research design quality. CONCLUSION: There is no evidence of the diagnostic value of QUS at the present time despite the overall excellent and adequate research design quality of primary studies. Although QUS can produce reliable measurements, insufficient evidence has been reported to support other clinimetric properties of this technique.

Recalcitrant intussusception: exploring potential associations with Helicobacter pylori infection - a case report and literature review.

BACKGROUND: Intussusception, a common cause of abdominal pain in children, often lacks clear underlying causes and is mostly idiopathic. Recurrence, though rare, raises clinical concerns, with rates escalating after each episode. Factors like pathological lead points and Henoch-Schönlein purpura (HSP) are associated with recurrent cases. On the other hand, the prevalence of Helicobacter pylori (H. pylori), often asymptomatic, in children has been declining. Although its infection is reported to be linked with HSP, its role in recurrent intussusception remains unexplored. Further research is needed to understand the interplay among H. pylori (culprit pathogen), HSP (trigger), and intractable intussusception so as to develop effective management strategies. CASE PRESENTATION: A two-year-old girl experienced four atypical episodes of intussusception at distinct locations, which later coincided with HSP. Despite treatment with steroids, recurrent intussusception persisted, suggesting that HSP itself was not a major cause for intractable presentations. Subsequent identification of H. pylori infection and treatment with triple therapy resulted in complete resolution of her recalcitrant intussusception. CONCLUSION: This instructive case underscored a sequence wherein H. pylori infection triggered HSP, subsequently resulting in recurrent intussusception. While H. pylori infection is not common in young children, the coexistence of intractable intussusception and steroid-resistant recurrent HSP necessitates consideration of H. pylori infection as a potential underlying pathogen.

Tunable Thermoelectric Performance of the Nanocomposites Formed by Diketopyrrolopyrrole/Isoindigo-Based Donor-Acceptor Random Conjugated Copolymers and Carbon Nanotubes.

This paper presents the development of thermoelectric properties in nanocomposites comprising donor-acceptor random conjugated copolymers and single-walled carbon nanotubes (SWCNTs). The composition of the conjugated polymers, specifically the ratio of diketopyrrolopyrrole (DPP) to isoindigo (IID), is manipulated to design a series of random conjugated copolymers (DPP0, DPP5, DPP10, DPP30, DPP50, DPP90, DPP95, and DPP100). The objective is to improve the dispersion of SWCNTs into smaller bundles, leading to enhanced thermoelectric properties of the polymer/SWCNT nanocomposite. This dispersion strategy promotes an interconnected conducting network, which plays a critical role in optimizing the thermoelectric performance. Accordingly, the effects of morphologies on the thermoelectric properties of the nanocomposites are systematically investigated. The DPP95/SWCNT nanocomposite exhibits the strongest interaction, resulting in the highest power factor (PF) of 711.1 µW m-1 K-2, derived from the high electrical conductivity of 1690 S cm-1 and Seebeck coefficient of 64.8 µV K-1. The prototype flexible thermoelectric generators assembled with a DPP95/SWCNT film achieve a maximum power output of 20.4 µW m-2 at a temperature difference of 29.3 K. These findings highlight the potential of manipulating the composition of random conjugated copolymers and incorporating SWCNTs to efficiently harvest low-grade waste heat in wearable thermoelectric devices.

Exploring CNS Involvement in Pain Insensitivity in Hereditary Sensory and Autonomic Neuropathy Type 4: Insights from Tc-99m ECD SPECT Imaging.

Hereditary sensory and autonomic neuropathy type 4 (HSAN4), also known as congenital insensitivity to pain with anhidrosis (CIPA), is a rare genetic disorder caused by NTRK1 gene mutations, affecting nerve growth factor signaling. This study investigates the central nervous system's (CNS) involvement and its relation to pain insensitivity in HSAN4. We present a 15-year-old girl with HSAN4, displaying clinical signs suggestive of CNS impact, including spasticity and a positive Babinski's sign. Using Technetium-99m ethyl cysteinate dimer single-photon emission computed tomography (Tc-99m ECD SPECT) imaging, we discovered perfusion deficits in key brain regions, notably the cerebellum, thalamus, and postcentral gyrus. These regions process pain signals, providing insights into HSAN4's pain insensitivity. This study represents the first visualization of CNS perfusion abnormality in an HSAN4 patient. It highlights the intricate relationship between the peripheral and central nervous systems in HSAN4. The complexity of HSAN4 diagnosis, involving potential unidentified genes, underscores the need for continued research to refine diagnostic approaches and develop comprehensive treatments.

The role of vascular smooth muscle cell membrane-bound thrombomodulin in neointima formation.

BACKGROUND AND AIMS: Thrombomodulin (TM) is an endothelial cell membrane-bound anticoagulant protein expressed in normal arteries. After vascular injury, medial and neointimal smooth muscle cells (SMCs) exhibit large amounts of TM. The purpose of this study was to investigate the physiological significance of vascular SMC-bound TM. METHODS: The morphology, expression of phenotype markers and cell behaviors of cultured aortic SMCs after knockdown of TM were observed. Transgenic mice with SMC-specific TM deletion were generated, and carotid neointima formation was induced by carotid ligation. RESULTS: Cultured human aortic SMCs displayed a synthetic phenotype with a rhomboid-shaped morphology and expressed TM. TM knockdown induced a spindle-shaped change in morphology with an increased expression of contractile phenotype marker and decreased expression of synthetic phenotype marker. TM knockdown not only attenuated the proliferation of SMCs but also reduced tumor necrosis factor-α-induced nuclear factor-κB activation and interlukin-6 production. In a carotid artery ligation model, transgenic mice with SMC-specific TM deletion (SM22-cretg/TMflox/flox) had significantly less cellular proliferation in arterial walls compared with wild type mice (SM22-cretg/TM+/+). The neointima area and neointima/media area ratio were smaller in SM22-cretg/TMflox/flox mice at 4 weeks after ligation. CONCLUSIONS: Our results indicate that vascular SMC-bound TM plays a role in changes of the SMC phenotype. It also influences SMC cell behavior and injury-induced neointima formation.

Tumor Endothelial Marker 1 (TEM1/Endosialin/CD248) Enhances Wound Healing by Interacting with Platelet-Derived Growth Factor Receptors.

Tumor endothelial marker 1 (TEM1), also known as endosialin or CD248, is a type I transmembrane glycoprotein containing a C-type lectin-like domain. It is highly expressed in pericytes and fibroblasts. Dermal fibroblasts play a pivotal role during cutaneous wound healing, especially in the proliferative phase. However, the physiological function of TEM1 in wound healing is still undetermined. During the process of wound healing, the expression of both TEM1 and platelet-derived growth factor (PDGF) receptor α was highly upregulated in myofibroblasts. In vivo, fibroblast activation and collagen deposition in granulation tissues were attenuated, and wound healing was retarded in TEM1-deleted mice. In vitro, the migration, adhesion, and proliferation of NIH3T3 cells were suppressed following TEM1 knockdown by short hairpin RNA. In PDGF-BB-treated NIH3T3 cells, the downstream signal and mitogenic, and chemoattractive effects were inhibited by TEM1 knockdown. In addition, TEM1 and PDGF receptor α were colocalized in subcellular organelles in fibroblasts, and the association of TEM1 and PDGF receptor α was demonstrated by coimmunoprecipitation. In summary, these findings suggested that TEM1, in combination with PDGF receptor α, plays a critical role in wound healing by enhancing the mitogenic and chemoattractive effects of PDGF-BB and collagen deposition in myofibroblasts.

Characterization and comparative studies of zebrafish and human recombinant dihydrofolate reductases--inhibition by folic acid and polyphenols.

Dihydrofolate reductase (DHFR) catalyzes folic acid reduction and recycles dihydrofolate generated during dTMP biosynthesis to tetrahydrofolate. DHFR is the main target of methotrexate, the most widely used agent for antifolate therapy. Nevertheless, the emergence of methotrexate-resistance has greatly impeded the curative potential of this drug. Therefore, drugs with improved efficacy are still in demand, as well as an efficient in vitro assay system and animal model for antifolate drug discovery. The aim of this study is to evaluate the suitability of using zebrafish DHFR as an alternative assay system for antifolate drug discovery. The cDNAs encoding zebrafish and human DHFR were cloned, overexpressed, and purified. Similar structural and kinetic properties were revealed between zebrafish and human recombinant DHFRs. The susceptibilities of both enzymes to known DHFR inhibitors, including methotrexate and trimethoprim, and compounds with antifolate potential, such as polyphenols, are also comparable. In addition, the DHFR-mediated dihydrofolate reduction was significantly inhibited by its own substrate folic acid. An unexpected tissue-specific distribution of DHFR was observed with the highest level present in ova and brains of zebrafish. DHFR is also abundant in zebrafish embryos of early stages and decreased abruptly after 3 days postfertilization. The substantial resemblance between zebrafish and human DHFRs, as demonstrated in this study, provides compelling evidence supporting the use of zebrafish DHFR as an in vitro assay system for folate-related studies and drug discovery.

Plasminogen/thrombomodulin signaling enhances VEGF expression to promote cutaneous wound healing.

Plasminogen (Plg) and thrombomodulin (TM) are glycoproteins well known for fibrinolytic and anticoagulant functions, respectively. Both Plg and TM are essential for wound healing. However, their significance during the reparative process was separately demonstrated in previous studies. Here, we investigate the interaction between Plg and epithelial TM and its effect on wound healing. Characterization of the wound margin revealed that Plg and TM were simultaneously upregulated at the early stage of wound healing and the two molecules were bound together. In vitro, TM silencing or knockout in keratinocytes inhibited Plg activation. Plg treatment enhanced keratinocyte proliferation and migration, and these actions were abolished by TM antibody. Keratinocyte-expressed vascular endothelial growth factor (VEGF), which presented a dose-response relationship with Plg treatment, can be suppressed by TM silencing. Moreover, treatment with VEGF antibody inhibited Plg-enhanced keratinocyte proliferation and wound recovery. In vivo, TM antibody treatment and keratinocyte-specific TM knockout can impede Plg-enhanced wound healing in mice. In high-glucose environments, Plg-enhanced VEGF expression and wound healing were suppressed due at least in part to downregulation of keratinocyte-expressed TM. Taken together, our findings suggest that activation of Plg/TM signaling may hold therapeutic potential for chronic wounds in diabetic or non-diabetic individuals. KEY MESSAGES: Plg binds to TM in cutaneous wound healing. TM facilitates the activation of Plg to Plm in keratinocytes. Epithelial TM regulates Plg-enhanced wound healing through VEGF expression.

Recombinant adeno-associated virus vector carrying the thrombomodulin lectin-like domain for the treatment of abdominal aortic aneurysm.

BACKGROUND AND AIMS: Thrombomodulin (TM), through its lectin-like domain (TMD1), sequesters proinflammatory high-mobility group box 1 (HMGB1) to prevent it from engaging the receptor for advanced glycation end product (RAGE) that sustains inflammation and tissue damage. Our previous study demonstrated that short-term treatment with recombinant TM containing all the extracellular domains (i.e., rTMD123) inhibits HMGB1-RAGE signaling and confers protection against CaCl2-induced AAA formation. In this study, we attempted to further optimize TM domains, as a potential therapeutic agent for AAA, using the recombinant adeno-associated virus (AAV) vector. METHODS: The therapeutic effects of recombinant TMD1 (rTMD1) and recombinant AAV vectors carrying the lectin-like domain of TM (rAAV-TMD1) were evaluated in the CaCl2-induced AAA model and angiotensin II-infused AAA model, respectively. RESULTS: In the CaCl2-induced model, treatment with rTMD1 suppressed the tissue levels of HMGB1 and RAGE, macrophage accumulation, elastin destruction and AAA formation, and the effects were comparable to a mole-equivalent dosage of rTMD123. In the angiotensin II-infused model, a single intravenous injection of rAAV-TMD1 (1011 genome copies), which resulted in a persistently high serum level of TMD1 for at least 12 weeks, effectively attenuated AAA formation with suppression of HMGB1 and RAGE levels and inhibition of proinflammatory cytokine production, macrophage accumulation, matrix metalloproteinase activities and oxidative stress in the aortic wall. CONCLUSIONS: These findings corroborate the therapeutic potential of the TM lectin-like domain in AAA. The attenuation of angiotensin II-infused AAA by one-time delivery of rAAV-TMD1 provides a proof-of-concept validation of its application as potential gene therapy for aneurysm development.

Recombinant Thrombomodulin Exerts Anti-autophagic Action in Endothelial Cells and Provides Anti-atherosclerosis Effect in Apolipoprotein E Deficient Mice.

Stress-induced alteration in endothelial cells (ECs) integrity precedes the development of atherosclerosis. Previous studies showed that the soluble recombinant thrombomodulin (rTM) not only increases ECs proliferation but also exerts anti-apoptotic activity in ECs. However, the functional significance of soluble rTM on autophagy-related apoptosis in ECs is still undetermined. Implicating a cytoprotective role for rTM in persistent serum starvation (SS)-induced autophagy in cultured ECs, we found that treatment of rTM decreased the expression of SS-induced autophagy-related proteins, ATG5 and LC3, and the formation of autophagosomes through activation of AKT/mTOR pathway. In addition, treatment of rTM decreased SS-induced EC apoptosis, but this effect of rTM could not be recapitulated by co-treatment with a potent autophagy inducer, rapamycin and in ECs with ATG5 knockdown. In human atherosclerosis specimens, expression of autophagy markers, ATG13 and LC3, were more abundant in aortic intimal ECs with severe atherosclerosis than those without atherosclerosis. Moreover, compared to saline treatment group, administration of rTM reduced LC3 and ATG13 expression, intimal EC apoptosis, and atherosclerotic lesion severity in the aorta of apolipoprotein E deficient mice. In conclusion, treatment with rTM suppressed stress-induced autophagy overactivation in ECs, provided ECs protective effects, and decreased atherosclerosis in apolipoprotein E deficient mice.

Thrombomodulin promotes focal adhesion kinase activation and contributes to angiogenesis by binding to fibronectin.

Angiogenesis promotes tumor growth and metastasis. Cell adhesion molecules interact with the extracellular matrix (ECM) and increase cell adhesion and migration during angiogenesis. Thrombomodulin (TM) is a cell surface transmembrane glycoprotein expressed in endothelial cells. However, the function and significance of TM in cell-matrix interactions and angiogenesis remain unclear. Here, we first demonstrated that recombinant lectin-like domain of TM interacts with an ECM protein, fibronectin, and identified the N-terminal 70-kDa domain of fibronectin as the TM-binding site. Exogenous expression of TM in TM-deficient A2058 melanoma cells enhanced cell adhesion and migration on fibronectin and invasion on Matrigel. In addition, TM increased focal adhesion kinase (FAK) phosphorylation and matrix metalloproteinase-9 production. In mice bearing subcutaneous B16F10 melanoma tumors, immunofluorescence analysis indicated that TM was highly expressed and co-localized with fibronectin on the tumor vasculature. The interaction between TM and fibronectin in tumor blood vessels was also validated by the proximity ligation assay. In human umbilical vein endothelial cells, up-regulation of TM by vascular endothelial growth factor (VEGF), a tumor angiogenic factor, promoted cell adhesion and tube formation, whereas TM knockdown by RNA interference attenuated VEGF-induced cell adhesion and tube formation. In summary, TM promotes angiogenesis by enhancing cell adhesion, migration, and FAK activation through interaction with fibronectin. TM may represent a novel target for inhibiting tumor angiogenesis.

Toll-Like Receptor 4 Is Essential in the Development of Abdominal Aortic Aneurysm.

Toll-like receptor (TLR) family plays a key role in innate immunity and various inflammatory responses. TLR4, one of the well-characterized pattern-recognition receptors, can be activated by endogenous damage-associated molecular pattern molecules such as high mobility group box 1 (HMGB1) to sustain sterile inflammation. Evidence suggested that blockade of TLR4 signaling may confer protection against abdominal aortic aneurysm (AAA). Herein we aimed to obtain further insight into the mechanism by which TLR4 might promote aneurysm formation. Characterization of the CaCl2-induced AAA model in mice revealed that upregulation of TLR4 expression, localized predominantly to vascular smooth muscle cells (VSMCs), was followed by a late decline during a 28-day period of AAA development. In vitro, TLR4 expression was increased in VSMCs treated with HMGB1. Knockdown of TLR4 by siRNA attenuated HMGB1-enhanced production of proinflammatory cytokines, specifically interleukin-6 and monocyte chemoattractant protein-1 (MCP-1), and matrix-degrading matrix metalloproteinase (MMP)-2 from VSMCs. In vivo, two different strains of TLR4-deficient (C57BL/10ScNJ and C3H/HeJ) mice were resistant to CaCl2-induced AAA formation compared to their respective controls (C57BL/10ScSnJ and C3H/HeN). Knockout of TLR4 reduced interleukin-6 and MCP-1 levels and HMGB1 expression, attenuated macrophage accumulation, and eventually suppressed MMP production, elastin destruction and VSMC loss. Finally, human AAA exhibited higher TLR4 expression that was localized to VSMCs. These data suggest that TLR4 signaling contributes to AAA formation by promoting a proinflammatory status of VSMCs and by inducing proteinase release from VSMCs during aneurysm initiation and development.

Peroxisome Proliferator-Activated Receptor γ Level Contributes to Structural Integrity and Component Production of Elastic Fibers in the Aorta.

Loss of integrity and massive disruption of elastic fibers are key features of abdominal aortic aneurysm (AAA). Peroxisome proliferator-activated receptor γ (PPARγ) has been shown to attenuate AAA through inhibition of inflammation and proteolytic degradation. However, its involvement in elastogenesis during AAA remains unclear. PPARγ was highly expressed in human AAA within all vascular cells, including inflammatory cells and fibroblasts. In the aortas of transgenic mice expressing PPARγ at 25% normal levels (Pparg(C) (/-) mice), we observed the fragmentation of elastic fibers and reduced expression of vital elastic fiber components of elastin and fibulin-5. These were not observed in mice with 50% normal PPARγ expression (Pparg(+/-) mice). Infusion of a moderate dose of angiotensin II (500 ng/kg per minute) did not induce AAA but Pparg(+/-) aorta developed flattened elastic lamellae, whereas Pparg(C/-) aorta showed severe destruction of elastic fibers. After infusion of angiotensin II at 1000 ng/kg per minute, 73% of Pparg(C/-) mice developed atypical suprarenal aortic aneurysms: superior mesenteric arteries were dilated with extensive collagen deposition in adventitia and infiltrations of inflammatory cells. Although matrix metalloproteinase inhibition by doxycycline somewhat attenuated the dilation of aneurysm, it did not reduce the incidence nor elastic lamella deterioration in angiotensin II-infused Pparg(C/-) mice. Furthermore, PPARγ antagonism downregulated elastin and fibulin-5 in fibroblasts, but not in vascular smooth muscle cells. Chromatin immunoprecipitation assay demonstrated PPARγ binding in the genomic sequence of fibulin-5 in fibroblasts. Our results underscore the importance of PPARγ in AAA development though orchestrating proper elastogenesis and preserving elastic fiber integrity.

Thrombomodulin promotes diabetic wound healing by regulating toll-like receptor 4 expression.

Keratinocyte-expressed thrombomodulin (TM) and the released soluble TM (sTM) have been demonstrated to promote wound healing. However, the effects of high glucose on TM expression in keratinocytes and the role of TM in diabetic ulcer remain unclear. In this study, we demonstrated that expressions of TM and Toll-like receptor 4 (TLR4) were both downregulated in high-glucose cultured human keratinocytes and in skin keratinocytes of diabetic patients. In addition, the wound-triggered upregulation of TM and sTM production was abolished in both high-glucose cultured human keratinocytes and streptozotocin-induced diabetic mouse skin. Furthermore, supplementation of recombinant sTM could increase TLR4 expression and promote cutaneous wound healing in both high-glucose cultured human keratinocytes and diabetic mice. However, in Tlr4-deleted mice, which exhibited delayed wound healing, the therapeutic benefit of recombinant sTM was abrogated. Moreover, our results showed that tumor necrosis factor-α (TNF-α) expression in keratinocytes was dose-dependently upregulated by glucose, and TNF-α treatment downregulated the expression of TM and TLR4. Taken together, high-glucose environment reduces the expression of TM and TLR4 in keratinocytes possibly through the action of TNF-α, and recombinant sTM can increase the TLR4 expression and promote wound healing under diabetic condition.

Thrombomodulin regulates keratinocyte differentiation and promotes wound healing.

The membrane glycoprotein thrombomodulin (TM) has been implicated in keratinocyte differentiation and wound healing, but its specific function remains undetermined. The epidermis-specific TM knockout mice were generated to investigate the function of TM in these biological processes. Primary cultured keratinocytes obtained from TM(lox/lox); K5-Cre mice, in which TM expression was abrogated, underwent abnormal differentiation in response to calcium induction. Poor epidermal differentiation, as evidenced by downregulation of the terminal differentiation markers loricrin and filaggrin, was observed in TM(lox/lox); K5-Cre mice. Silencing TM expression in human epithelial cells impaired calcium-induced extracellular signal-regulated kinase pathway activation and subsequent keratinocyte differentiation. Compared with wild-type mice, the cell spreading area and wound closure rate were lower in keratinocytes from TM(lox/lox); K5-Cre mice. In addition, the lower density of neovascularization and smaller area of hyperproliferative epithelium contributed to slower wound healing in TM(lox/lox); K5-Cre mice than in wild-type mice. Local administration of recombinant TM (rTM) accelerated healing rates in the TM-null skin. These data suggest that TM has a critical role in skin differentiation and wound healing. Furthermore, rTM may hold therapeutic potential for the treatment of nonhealing chronic wounds.

The epidermal growth factor-like domain of CD93 is a potent angiogenic factor.

Human CD93, an epidermal growth factor (EGF)-like domain containing transmembrane protein, is predominantly expressed in the vascular endothelium. Studies have shown that AA4, the homolog of CD93 in mice, may mediate cell migration and angiogenesis in endothelial cells. Soluble CD93 has been detected in the plasma of healthy individuals. However, the role of soluble CD93 in the endothelium remains unclear. Recombinant soluble CD93 proteins with EGF-like domains (rCD93D123, with domains 1, 2, and 3; and rCD93D23, with domains 2 and 3) were generated to determine their functions in angiogenesis. We found that rCD93D23 was more potent than rCD93D123 in stimulating the proliferation and migration of human umbilical vein endothelial cells (HUVECs). Production of matrix-metalloproteinase 2 increased after the HUVECs were treated with rCD93D23. Further, in a tube formation assay, rCD93D23 induced cell differentiation of HUVECs through phosphoinositide 3-kinase/Akt/endothelial nitric oxide synthase and extracellular signal-regulated kinases-1/2 signaling. Moreover, rCD93D23 promoted blood vessel formation in a Matrigel-plug assay and an oxygen-induced retinopathy model in vivo. Our findings suggest that the soluble EGF-like domain containing CD93 protein is a novel angiogenic factor acting on the endothelium.