[Effect of Erchen Decoction on liver mitochondrial function by inhibiting mTORC1/SREBP1/CAV1 pathway in mice with high-fat diet].

This study aims to investigate the effect of Erchen Decoction(ECD) on liver mitochondrial function in mice with a high-fat diet and its possible mechanism. A total of sixty C57BL/6J mice were randomly divided into a normal group, high-fat group, ECD group, mTORC1 activator(MHY) group, ECD+MHY group, and polyene phosphatidyl choline(PPC) group, with 10 rats in each group. The normal group was given a normal diet, and the other groups were fed a high-fat diet for 20 weeks. At the 17th week, the ECD group and ECD+MHY group were given ECD(8.7 g·kg~(-1)) daily, and the PPC group was given PPC(0.18 g·kg~(-1)) daily, while the remaining groups were given normal saline(0.01 mL·g~(-1)) daily for four weeks. In the 19th week, the MHY group and ECD+MHY group were injected intraperitoneally with MHY(5 mg·kg~(-1)) every other day for two weeks. During the experiment, the general conditions of the mice were observed. The contents of triglyceride(TG) and total cholesterol(TC) in serum were measured. Morphological changes in liver tissue were examined through HE and oil red O staining. The content of adenosine triphosphate(ATP) was determined using chemiluminescence, and mitochondrial membrane potential was assessed using a fluorescence probe(JC-1). Western blot was performed to detect the expression of rapamycin target protein complex 1(mTOR1), ribosomal protein S6 kinase B1(S6K), sterol regulatory element binding protein 1(SREBP1), and caveolin 1(CAV1). RESULTS:: revealed that compared with the normal group, the mice in the high-fat group exhibited significant increases in body weight and abdominal circumference(P<0.01). Additionally, there were significant increases in TG and TC levels(P<0.01). HE and oil red O staining showed that the boundaries of hepatic lobules were unclear; hepatocytes were enlarged, round, and irregularly arranged, with obvious lipid droplet deposition and inflammatory cell infiltration. The liver ATP content and mitochondrial membrane potential decreased significantly(P<0.01). The expression of p-mTOR, p-S6K, and n-SREBP1 increased significantly(P<0.01), while the expression of CAV1 decreased significantly(P<0.01). Compared with the high-fat group, the body weight and TG content of mice in the ECD group and PPC group decreased significantly(P<0.05). Improvements were observed in hepatocyte morphology, lipid deposition, and inflammatory cell infiltration. Furthermore, there were significant increases in ATP content and mitochondrial membrane potential(P<0.05 or P<0.01). The expression of p-mTOR, p-S6K, and n-SREBP1 decreased significantly in the ECD group(P<0.01), while CAV1 expression increased significantly(P<0.01). However, the indices mentioned above did not show improvement in the MHY group. When the ECD+MHY group was compared with the MHY group, there were significant reductions in body weight and TG contents(P<0.05). The morphological changes of hepatocytes, lipid deposition, and inflammatory cell infiltration were recovered. Moreover, there were significant increases in liver ATP content and mitochondrial membrane potential(P<0.05 or P<0.05). The expression of p-mTOR, p-S6K, and n-SREBP1 decreased significantly(P<0.01), while CAV1 expression increased significantly(P<0.01). In conclusion, ECD can improve mitochondrial function by regulating the mTORC1/SREBP1/CAV1 pathway. This mechanism may be involved in the resolution of phlegm syndrome and the regulation of lipid metabolism.

DHCR24 Insufficiency Promotes Vascular Endothelial Cell Senescence and Endothelial Dysfunction via Inhibition of Caveolin-1/ERK Signaling.

Endothelial cells (ECs) senescence is critical for vascular dysfunction, which leads to age-related disease. DHCR24, a 3ß-hydroxysterol δ 24 reductase with multiple functions other than enzymatic activity, has been involved in age-related disease. However, little is known about the relationship between DHCR24 and vascular ECs senescence. We revealed that DHCR24 expression is chronologically decreased in senescent human umbilical vein endothelial cells (HUVECs) and the aortas of aged mice. ECs senescence in endothelium-specific DHCR24 knockout mice was characterized by increased P16 and senescence-associated secretory phenotype, decreased SIRT1 and cell proliferation, impaired endothelium-dependent relaxation, and elevated blood pressure. In vitro, DHCR24 knockdown in young HUVECs resulted in a similar senescence phenotype. DHCR24 deficiency impaired endothelial migration and tube formation and reduced nitric oxide (NO) levels. DHCR24 suppression also inhibited the caveolin-1/ERK signaling, probably responsible for increased reactive oxygen species production and decreased eNOS/NO. Conversely, DHCR24 overexpression enhanced this signaling pathway, blunted the senescence phenotype, and improved cellular function in senescent cells, effectively blocked by the ERK inhibitor U0126. Moreover, desmosterol accumulation induced by DHCR24 deficiency promoted HUVECs senescence and inhibited caveolin-1/ERK signaling. Our findings demonstrate that DHCR24 is essential in ECs senescence.

EPIC-1042 as a potent PTRF/Cavin1-caveolin-1 interaction inhibitor to induce PARP1 autophagic degradation and suppress temozolomide efflux for glioblastoma.

BACKGROUND: Temozolomide (TMZ) treatment efficacy in glioblastoma is determined by various mechanisms such as TMZ efflux, autophagy, base excision repair (BER) pathway, and the level of O6-methylguanine-DNA methyltransferase (MGMT). Here, we reported a novel small-molecular inhibitor (SMI) EPIC-1042 (C20H28N6) with the potential to decrease TMZ efflux and promote PARP1 degradation via autolysosomes in the early stage. METHODS: EPIC-1042 was obtained from receptor-based virtual screening. Co-immunoprecipitation and pull-down assays were applied to verify the blocking effect of EPIC-1042. Western blotting, co-immunoprecipitation, and immunofluorescence were used to elucidate the underlying mechanisms of EPIC-1042. In vivo experiments were performed to verify the efficacy of EPIC-1042 in sensitizing glioblastoma cells to TMZ. RESULTS: EPIC-1042 physically interrupted the interaction of PTRF/Cavin1 and caveolin-1, leading to reduced secretion of small extracellular vesicles (sEVs) to decrease TMZ efflux. It also induced PARP1 autophagic degradation via increased p62 expression that more p62 bound to PARP1 and specially promoted PARP1 translocation into autolysosomes for degradation in the early stage. Moreover, EPIC-1042 inhibited autophagy flux at last. The application of EPIC-1042 enhanced TMZ efficacy in glioblastoma in vivo. CONCLUSION: EPIC-1042 reinforced the effect of TMZ by preventing TMZ efflux, inducing PARP1 degradation via autolysosomes to perturb the BER pathway and recruitment of MGMT, and inhibiting autophagy flux in the later stage. Therefore, this study provided a novel therapeutic strategy using the combination of TMZ with EPIC-1042 for glioblastoma treatment.

Caveolin-1-derived peptide attenuates cigarette smoke-induced airway and alveolar epithelial injury.

Chronic obstructive pulmonary disease (COPD) is a debilitating lung disease with no effective treatment that can reduce mortality or slow the disease progression. COPD is the third leading cause of global death and is characterized by airflow limitations due to chronic bronchitis and alveolar damage/emphysema. Chronic cigarette smoke (CS) exposure damages airway and alveolar epithelium and remains a major risk factor for the pathogenesis of COPD. We found that the expression of caveolin-1, a tumor suppressor protein; p53; and plasminogen activator inhibitor-1 (PAI-1), one of the downstream targets of p53, was markedly increased in airway epithelial cells (AECs) as well as in type II alveolar epithelial (AT2) cells from the lungs of patients with COPD or wild-type mice with CS-induced lung injury (CS-LI). Moreover, p53- and PAI-1-deficient mice resisted CS-LI. Furthermore, treatment of AECs, AT2 cells, or lung tissue slices from patients with COPD or mice with CS-LI with a seven amino acid caveolin-1 scaffolding domain peptide (CSP7) reduced mucus hypersecretion in AECs and improved AT2 cell viability. Notably, induction of PAI-1 expression via increased caveolin-1 and p53 contributed to mucous cell metaplasia and mucus hypersecretion in AECs, and reduced AT2 viability, due to increased senescence and apoptosis, which was abrogated by CSP7. In addition, treatment of wild-type mice having CS-LI with CSP7 by intraperitoneal injection or nebulization via airways attenuated mucus hypersecretion, alveolar injury, and significantly improved lung function. This study validates the potential therapeutic role of CSP7 for treating CS-LI and COPD. NEW & NOTEWORTHY Chronic cigarette smoke (CS) exposure remains a major risk factor for the pathogenesis of COPD, a debilitating disease with no effective treatment. Increased caveolin-1 mediated induction of p53 and downstream plasminogen activator inhibitor-1 (PAI-1) expression contributes to CS-induced airway mucus hypersecretion and alveolar wall damage. This is reversed by caveolin-1 scaffolding domain peptide (CSP7) in preclinical models, suggesting the therapeutic potential of CSP7 for treating CS-induced lung injury (CS-LI) and COPD.

Interleukin-37 inhibits desmoglein-3 endocytosis and keratinocyte dissociation via upregulation of Caveolin-1 and inhibition of the STAT3 pathway.

BACKGROUND: Pemphigus vulgaris (PV) is a potentially fatal autoimmune bullous disease primarily caused by acantholysis of keratinocytes attributed to pathogenic desmoglein-3 (Dsg3) autoantibodies. Interleukin-37 (IL-37) reportedly plays important roles in a variety of autoimmune diseases, but its role in PV is not clear. OBJECTIVES: To investigate whether IL-37 plays a role in the occurrence and progression of PV. METHODS: HaCaT keratinocytes were stimulated with anti-Dsg3 antibody to establish an in vitro PV model, which was defined as anti-Dsg3 group. Cells incubated with medium without anti-Dsg3 treatment were used as control. IL-37 was cultured with these cells infected with or without lentiviral vector shRNA-Caveolin-1 (sh-Cav-1-LV). Cell dissociation assay and immunocytofluorescence were performed to assess keratinocyte dissociation, keratin retraction and Dsg3 endocytosis. Real-time PCR was used to detect the mRNA level of Cav-1, and western blot was used to determine the protein expression of Cav-1, Dsg3, STAT3 and phosphorylated-STAT3 (p-STAT3). RESULTS: The anti-Dsg3 group showed more cell debris, increased keratin retraction, increased Dsg3 endocytosis, reduced Cav-1 expression and co-localization than the control group, while IL-37 treatment neutralized all of these changes. Interestingly, Cav-1 knockdown supressed the inhibitory effect of IL-37 on keratinocyte dissociation and Dsg3 internalization. The protein expression of p-STAT3 was increased in keratinocytes of the PV model but decreased by IL-37. Re-activation of the STAT3 pathway by colivelin supressed the inhibitory effect of IL-37 on keratinocyte dissociation and Dsg3 internalization, along with upregulation of Cav-1 and Dsg3. CONCLUSIONS: IL-37 inhibited keratinocyte dissociation and Dsg3 endocytosis in an in vitro PV model through the upregulating Cav-1 and inhibiting STAT3 pathway.

Serum amyloid A and interleukin -1ß facilitate LDL transcytosis across endothelial cells and atherosclerosis via NF-κB/caveolin-1/cavin-1 pathway.

BACKGROUND AND AIMS: Inflammatory molecules play important roles in atherosclerosis. We aimed to illustrate the roles of serum amyloid A (SAA), and interleukin (IL)-1ß in low density lipoproteins (LDL) transcytosis and atherosclerosis. METHODS: Effects of SAA and IL-1ß on transcytosis of LDL were measured by an in vitro LDL transcytosis model. NF-κB/caveolin-1/cavin-1 pathway activation was investigated by Western blots and ELISA. Effects of SAA and IL-1ß on the retention of LDL in aorta of C57BL/6J mice were detected by IVIS spectrum. Effects of SAA and IL-1ß on atherosclerosis in Apoe-/- mice were examined by Oil Red O staining. RESULTS: SAA and IL-1ß stimulated LDL transcytosis across endothelial cells (ECs), which was accompanied by an increase in LDL uptake by ECs. SAA and IL-1ß enhanced the activity of nuclear factor (NF)-κB, consequently facilitating an up-regulation of proteins involved in caveolae formation, including caveolin-1 and cavin-1, along with an assembly of NLRP3 inflammasome. Furthermore, SAA- and IL-1ß-induced effects were blocked by NF-κB subunit p65 siRNA. Meanwhile, SAA- and IL-1ß-induced LDL transcytosis were effectively blocked by caveolin-1 siRNA or cavin-1 siRNA. Interestingly, SAA and IL-1ß facilitated LDL entering into the aorta of C57BL/6J mice. In Apoe-/- mice, SAA and IL-1ß increased the areas of lipid-rich atherosclerotic lesions in the both ascending and root of aorta. Furthermore, a significant increase in the NLRP3 inflammasome, accompanied by accumulation of cavin-1 and caveolin-1, was observed in the aortic endothelium of Apoe-/- mice. CONCLUSIONS: SAA and IL-1ß accelerated LDL transcytosis via the NF-κB/caveolin-1/cavin-1 axis.

Therapeutic potential of amitriptyline for paraquat-induced pulmonary fibrosis: Involvement of caveolin-1-mediated anti-epithelial-mesenchymal transition and inhibition of apoptosis.

OBJECTIVE: Treatment of pulmonary fibrosis caused by paraquat (PQ) poisoning remains problematic. Amitriptyline (AMT) has multiple pharmacological effects. Here we investigated the anti-fibrotic effect of AMT on PQ-induced pulmonary fibrosis and its possible mechanism. METHODS: C57BL/6 mice were randomly divided into control, PQ, PQ + AMT and AMT groups. Histopathology of the lungs, blood gas analysis, and levels of hydroxyproline (HYP), transforming growth factor ß1 (TGF-ß1) and interleukin 17 (IL-17) were measured. The siRNA transfection inhibited caveolin-1 in A549 cells, which induced epithelial-mesenchymal transition (EMT) by PQ and followed intervention with AMT. E-cadherin, N-cadherin, α-smooth muscle actin (α-SMA) and caveolin-1 were studied by immunohistochemistry and western blot analysis. The apoptosis rate was measured by flow cytometry. RESULTS: Compared with the PQ group, the PQ + AMT group displayed mild pathological changes in pulmonary fibrosis, lower HYP, IL-17 and TGF- ß1 levels in lung, but high TGF- ß1 in serum. Levels of N-cadherin and α-SMA in the lungs were significantly decreased, but caveolin-1 was increased, while SaO2 and PaO2 levels were higher. Compared with the PQ group, the apoptosis rate, N-cadherin and α-SMA levels in A549 cells were significantly decreased after PQ treatment and high dose AMT intervention (p < 0.01). The expressions of E-cadherin, N-cadherin and α-SMA in the PQ-induced cells transfected with caveolin-1 siRNA or siControl RNA were significantly different (p < 0.01), but the apoptosis rate was unaltered. CONCLUSION: AMT inhibited PQ-induced EMT in A549 cells and improved lung histopathology and oxygenation in mice by up-regulating caveolin-1.

Crucial Function of Caveolin-1 in Deoxynivalenol-Induced Enterotoxicity by Activating ROS-Dependent NLRP3 Inflammasome-Mediated Pyroptosis.

Deoxynivalenol (DON) is one of the most pervasive contaminating mycotoxins in grain, and exposure to DON is known to cause acute and chronic intestinal damage. As the gut is the most important target organ of DON, it is essential to identify the pivotal molecules involved in DON-induced enterotoxicity as well as the potential regulatory mechanisms. In the present study, we found that DON treatment dramatically decreased the jejunal villus height and increased the crypt depth in mice. DON exposure induced oxidative stress and NLRP3 inflammasome activation while increasing the levels of pyroptosis-related factors GSDMD, ASC, Caspase-1 P20, and IL-1ß and inflammatory cytokines IL-18, TNF-α, and IL-6. In vitro, 0.5-2 µM DON caused cytotoxicity and oxidative stress, as well as NLRP3-mediated pyroptosis in IPEC-J2 cells. Furthermore, DON treatment substantially improved the expression of Caveolin-1 (Cav-1) in vitro and in vivo. Interestingly, Cav-1 knockdown effectively attenuated DON-induced oxidative stress and NLRP3-mediated pyroptosis in IPEC-J2 cells. Meanwhile, treatment with the antioxidant NAC significantly alleviated DON-induced cytotoxicity and pyroptosis in IPEC-J2 cells. Likewise, after inhibiting NLRP3 inflammasome activation with the inhibitor MCC950, DON-induced cytotoxicity, pyroptosis, and inflammatory response were attenuated. However, NLRP3 inhibition did not affect Cav-1 expression. In conclusion, our study demonstrated that pyroptosis may be an underlying mechanism in DON-induced intestinal injury, and Cav-1 plays a pivotal role in DON-induced pyroptosis via regulating oxidative stress, which suggests a novel strategy to overcome DON-induced enterotoxicity.

Adipose tissue-derived mesenchymal stem cells attenuate lung inflammation and fibrosis in the bleomycin-induced pulmonary fibrosis rat model via caveolin-1/NF-kB signaling axis.

Stem cells have emerged as promising therapeutic options for several human diseases, including pulmonary fibrosis (PF). In this study, we investigated the therapeutic effects of adipose tissue-derived mesenchymal stem cells (ADMSCs) in the bleomycin-induced PF model rats and the underlying mechanisms. The PF model rats were generated by intratracheal injections of 5 mg/kg bleomycin sulfate. The ADMSC group rats were generated by injecting 2×10(6) ADMSCs via the tail vein at 0, 12, and 24 h after bleomycin injection. The control, PF, and ADMSC group rats were sacrificed on day 21 after bleomycin injections and the changes in lung histology and the levels of pro-inflammatory cytokines, collagen I, and caveolin-1 (Cav-1), and the activity of the NF-kappaB signaling pathway in the lung tissues was assessed by hematoxylin-eosin staining, ELISA, and western blotting assays. The lung tissues of the PF model rats showed significant infiltration of neutrophils, tissue destruction, and collagen deposition, but these effects were abrogated by the ADMSCs. The levels of pro-inflammatory cytokines such as IL-6, IL-1beta, and TGF-beta1 were elevated in the lung tissues and the bronchoalveolar lavage fluid (BALF) of the bleomycin-induced PF model rats, but these effects were reversed by the ADMSCs. The lung tissues of the PF model rats showed significant downregulation of Cav-1 and significantly higher activation of the pro-inflammatory NF-kappaB pathway. However, administration of the ADMSCs restored the expression levels of Cav-1 and suppressed the NF-kappaB signaling pathway in the lungs of the bleomycin-induced PF model rats. In conclusion, this study demonstrated that the ADMSCs protected against bleomycin-induced PF in the rat model by modulating the Cav-1/NF-kappaB axis.

Abnormal inflammatory traits and downregulated caveolin-1 expression in monocytes of psoriasis patients may be associated with psoriatic inflammation and atherosclerosis.

BACKGROUND: Monocytes and macrophages are implicated in inflammation and atherosclerosis, whereas monocytes are involved in psoriasis lesion formation. We previously reported a psoriatic inflammation-associated significant decrease in the membrane protein caveolin-1 (CAV-1) in psoriasis patient monocytes. However, the phenotype of circulating monocytes and their macrophage differentiation in psoriasis patients remain unclear. OBJECTIVE: We sought to clarify circulating monocyte and monocyte-derived macrophage (MDM) phenotypes in psoriasis patients with and without comorbidities. METHODS: Thirty-one patients with psoriasis vulgaris and 28 control subjects were included. Surface macrophage markers and inflammatory status were examined in circulating monocytes and MDMs from both groups. Expression of CD36, which mediates macrophage uptake of oxidized low-density lipoprotein (oxLDL), was evaluated in these cells. CAV-1-silenced monocytes were differentiated into macrophages to investigate the effects of CAV-1 downregulation on psoriatic inflammation and atherosclerosis. RESULTS: Macrophage surface markers were detectable in circulating monocytes. A significant M1 shift was detected in monocytes and MDMs in psoriasis patients, including those without cardiovascular disease risk factors, as compared to controls. MDMs of psoriasis patients had more CD36-expressing cells, which are associated with atherosclerosis risk. Additionally, CAV-1-silencing in monocytes increased the likelihood of M1-biased macrophage differentiation and increased pro-inflammatory cytokine production. CONCLUSIONS: Monocytes from psoriasis patients were more likely to differentiate into M1-dominant macrophages, correlating with inflammatory status and CAV-1 expression. These aberrant inflammatory monocytes not only contribute to psoriatic inflammation by producing psoriatic cytokines, but also have a phenotype that could increase atherosclerosis risk by uptake of oxLDL and formation of foam cells.

Melanosomal targeting via caveolin-1 dependent endocytosis mediates ZN(II) phthalocyanine phototoxic action in melanoma cells.

Melanosomes have been considered crucial targets in melanoma treatments. In this study we explored the role of melanosomes in photodynamic therapy (PDT), employing the synthetic Zn(II) phthalocyanine Pc13, a potent photosensitizer that promotes melanoma cell death after irradiation. Phototoxic action is mediated by reactive oxygen species increase. The internalization mechanism of Pc13 and its consequent subcellular localization were evaluated in melanotic B16-F0 cells. Pharmacological inhibitors of dynamin or caveolae, but not of clathrin, decreased Pc13 cellular uptake and phototoxicity. Similar results were obtained when cells over-expressed dominant negative mutants of dynamin-2 and caveolin-1, indicating that Pc13 is internalized by caveolae-mediated endocytosis. Confocal microscopy analysis revealed that Pc13 targets melanosomes and damage of these structures after irradiation was demonstrated by transmission electron microscopy. Treatment of pigmented B16-F0 and WM35 melanoma cells with the melanin synthesis inhibitor phenylthiourea for 48 h led to cell depigmentation and enhanced cell death after irradiation, whereas a 3-h period of inhibition did not modify melanin content but produced a marked reduction of Pc13 phototoxicity, together with a decrease of oxidative melanin synthesis intermediates. In contrast, the effect of Pc13 in amelanotic A375 cells was not altered by phenylthiourea treatment. These results provide evidence that melanosomes have a dual role in the efficacy of PDT. While melanin antagonizes the phototoxic action of Pc13, the release of cytotoxic synthetic intermediates to cytosol after irradiation and melanosome damage is conducive to the phototoxic response. Based on these findings, we demonstrate that melanosome-targeted PDT could be an effective approach for melanoma treatment.

Docosahexaenoic Acid Ameliorates the Toll-Like Receptor 22-Triggered Inflammation in Fish by Disrupting Lipid Raft Formation.

BACKGROUND: Although dietary DHA alleviates Toll-like receptor (TLR)-associated chronic inflammation in fish, the underlying mechanism is not well understood. OBJECTIVES: This study aimed to explore the role of Tlr22 in the innate immunity of large yellow croaker and investigate the anti-inflammatory effects of DHA on Tlr22-triggered inflammation. METHODS: Head kidney-derived macrophages of croaker and HEK293T cells were or were not pretreated with 100 µM DHA for 10 h prior to polyinosinic-polycytidylic acid (poly I:C) stimulation. We executed qRT-PCR, immunoblotting, and lipidomic analysis to examine the impact of DHA on Tlr22-triggered inflammation and membrane lipid composition. In vivo, croakers (12.03 ± 0.05 g) were fed diets containing 0.2% [control (Ctrl)], 0.8%, and 1.6% DHA for 8 wk before injection with poly I:C. Inflammatory genes expression and rafts-related lipids and protein expression were measured in the head kidney. Data were analyzed by ANOVA or Student t test. RESULTS: The activation of Tlr22 by poly I:C induced inflammation, and DHA diminished Tlr22-targeted inflammatory gene expression by 56-73% (P ≤ 0.05). DHA reduced membrane sphingomyelin (SM) and SFA-containing phosphatidylcholine (SFA-PC) contents, as well as lipid raft marker caveolin 1 amounts. Furthermore, lipid raft disruption suppressed Tlr22-induced Nf-κb and interferon h activation and p65 nuclear translocation. In vivo, expression of Tlr22 target inflammatory genes was 32-64% lower in the 1.6% DHA group than in the Ctrl group upon poly I:C injection (P ≤ 0.05). Also, the 1.6% DHA group showed a reduction in membrane SM and SFA-PC contents, accompanied by a decrease in caveolin 1 amounts, compared with the Ctrl group. CONCLUSIONS: The activation of Tlr22 signaling depends on lipid rafts, and DHA ameliorates the Tlr22-triggered inflammation in both head kidney and head kidney-derived macrophages of croaker partially by altering membrane SMs and SFA-PCs that are required for lipid raft organization.

Stabilizing and upregulating Axin with tankyrase inhibitor reverses 5-fluorouracil chemoresistance and proliferation by targeting the WNT/caveolin-1 axis in colorectal cancer cells.

Chemoresistance is a main obstacle for colorectal cancer treatment. In this study, we evaluated the effects and mechanisms of the WNT/ß-catenin signaling pathway on the chemoresistance of SW480 and SW620 colorectal cancer cells. The activity of ß-catenin was activated/inhibited by the small molecule compound GSK-3 inhibitor 6-bromo-indirubin-3'-oxime and the tankyrase inhibitor XAV939. The downstream target genes of the WNT/ß-catenin signaling pathway were screened using a cDNA microarray and bioinformatics analysis. Apoptosis induced by 5-Fu, cell cycle distribution and expression levels of WNT/ß-catenin/TCF12/caveolin-1 and multidrug resistance proteins were examed by flow cytometry and western blot after ß-catenin activation/inhibition and caveolin-1 overexpression/interference. The effect and mechanism of XAV939 on proliferation and apoptosis induced by 5-Fu in xenograft tumors of nude mice were evaluated by immunohistochemistry and TUNEL staining. 6-Bromo-indirubin-3'-oxime treatment increased ß-catenin expression by regulating GSK-3ß phosphorylation, accompanied by upregulation of TCF12, caveolin-1, P-gp, and MRP2 and downregulation of apoptosis induced by 5-Fu. Conversely, XAV939 treatment decreased ß-catenin expression by upregulating Axin, accompanied by downregulation of TCF12, Caveolin-1, P-gp, and MRP2 and upregulation of apoptosis induced by 5-Fu. The caveolin-1 gene was identified as an important downstream gene of the WNT/ß-catenin signaling pathway. Caveolin-1 overexpression upregulated ß-catenin expression, increased P-gp and MRP2 expression and decreased apoptosis induced by 5-Fu; conversely, caveolin-1 interference caused the opposite effects. In addition, in vivo experiments showed that XAV939 treatment reduced ß-catenin expression, increased apoptosis induced by 5-Fu and repressed xenograft tumor growth. Our findings suggested that inhibition of WNT/ß-catenin/TCF12/caveolin-1 provides a new promising therapeutic strategy for colorectal cancer treatment.

Regulation of Myogenesis by a Na/K-ATPase α1 Caveolin-Binding Motif.

The N-terminal caveolin-binding motif (CBM) in Na/K-ATPase (NKA) α1 subunit is essential for cell signaling and somitogenesis in animals. To further investigate the molecular mechanism, we have generated CBM mutant human-induced pluripotent stem cells (iPSCs) through CRISPR/Cas9 genome editing and examined their ability to differentiate into skeletal muscle (Skm) cells. Compared with the parental wild-type human iPSCs, the CBM mutant cells lost their ability of Skm differentiation, which was evidenced by the absence of spontaneous cell contraction, marker gene expression, and subcellular myofiber banding structures in the final differentiated induced Skm cells. Another NKA functional mutant, A420P, which lacks NKA/Src signaling function, did not produce a similar defect. Indeed, A420P mutant iPSCs retained intact pluripotency and ability of Skm differentiation. Mechanistically, the myogenic transcription factor MYOD was greatly suppressed by the CBM mutation. Overexpression of a mouse Myod cDNA through lentiviral delivery restored the CBM mutant cells' ability to differentiate into Skm. Upstream of MYOD, Wnt signaling was demonstrated from the TOPFlash assay to have a similar inhibition. This effect on Wnt activity was further confirmed functionally by defective induction of the presomitic mesoderm marker genes BRACHYURY (T) and MESOGENIN1 (MSGN1) by Wnt3a ligand or the GSK3 inhibitor/Wnt pathway activator CHIR. Further investigation through immunofluorescence imaging and cell fractionation revealed a shifted membrane localization of ß-catenin in CBM mutant iPSCs, revealing a novel molecular component of NKA-Wnt regulation. This study sheds light on a genetic regulation of myogenesis through the CBM of NKA and control of Wnt/ß-catenin signaling.

The Caveolin-1 Scaffolding Domain Peptide Reverses Aging-Associated Deleterious Changes in Multiple Organs.

Aging is a progressive, multifactorial, degenerative process in which deleterious changes occur in the biochemistry and function of organs. We showed that angiotensin II (AngII)-induced pathologies in the heart and kidney of young (3-month-old) mice are suppressed by the caveolin-1 scaffolding domain (CSD) peptide. Because AngII mediates many aging-associated changes, we explored whether CSD could reverse pre-existing pathologies and improve organ function in aged mice. Using 18-month-old mice (similar to 60-year-old humans), we found that >5-fold increases in leakage of serum proteins and >2-fold increases in fibrosis are associated with aging in the heart, kidney, and brain. Because tyrosine phosphorylation of cell junction proteins leads to the loss of microvascular barrier function, we analyzed the activation of the receptor tyrosine kinase PDGFR and the nonreceptor tyrosine kinases c-Src and Pyk2. We observed 5-fold activation of PDGFR and 2- to 3-fold activation of c-Src and Pyk2 in aged mice. Treatment with CSD for 4 weeks reversed these pathologic changes (microvascular leakage, fibrosis, kinase activation) in all organs almost down to the levels in healthy, young mice. In studies of heart function, CSD reduced the aging-associated increase in cardiomyocyte cross-sectional area and enhanced ventricular compliance in that echocardiographic studies demonstrated improved ejection fraction and fractional shortening and reduced isovolumic relation time. These results suggest that versions of CSD may be developed as treatments for aging-associated diseases in human patients based on the concept that CSD inhibits tyrosine kinases, leading to the inhibition of microvascular leakage and associated fibrosis, thereby improving organ function. SIGNIFICANCE STATEMENT: The caveolin-1 scaffolding domain (CSD) peptide reverses aging-associated fibrosis, microvascular leakage, and organ dysfunction in the heart, kidneys, and brain via a mechanism that involves the suppression of the activity of multiple tyrosine kinases, suggesting that CSD can be developed as a treatment for a wide range of diseases found primarily in the aged.

Localized Delivery of Caveolin-1 Peptide Assisted by Ultrasound-Mediated Microbubble Destruction Potentiates the Inhibition of Nitric Oxide-Dependent Vasodilation Response.

In the endothelium, nitric oxide synthase (eNOS) is the enzyme that generates nitric oxide, a key molecule involved in a variety of biological functions and cancer-related events. Therefore, selective inhibition of eNOS represents an attractive therapeutic approach for NO-related diseases and anticancer therapy. Ultrasound-mediated microbubble destruction (UMMD) conjugated with cell-permeable peptides has been investigated as a drug delivery system for effective delivery of anticancer molecules. We investigated the feasibility of loading antennapedia-caveolin-1 peptide (AP-Cav), a specific eNOS inhibitor, onto microbubbles to be delivered by UMMD in rat aortic endothelium. AP-Cav-loaded microbubbles (AP-Cav-MBs) and US parameters were characterized. Aortas were treated with UMMD for 30 s with 1.3 × 108 MBs/mL AP-Cav (8 µM)-MBs at 100-Hz pulse repetition frequency, 0.5-MPa acoustic pressure, 0.5 mechanical index and 10% duty cycle. NO-dependent vascular responses were assessed using an isolated organ system, 21 h post-treatment. Maximal relaxation response was inhibited 61.8% ± 1.6% in aortas treated with UMMD-AP-Cav-MBs, while in aortas treated with previously disrupted AP-Cav-MBs and then US, the inhibition was 31.6% ± 1.6%. The vascular contractile response was not affected. The impact of UMMD was evaluated in aortas treated with free AP-Cav; 30 µM of free AP-Cav was necessary to reach an inhibition response similar to that obtained with UMMD-AP-Cav-MBs. In conclusion, UMMD enhances the delivery and potentiates the effect of AP-Cav in the endothelial layer of rat aorta segments.

Caveolin-1 Promotes Cellular Senescence in Exchange for Blocking Subretinal Fibrosis in Age-Related Macular Degeneration.

Purpose: To determine whether caveolin-1 (i) prevents epithelial-mesenchymal transition in the RPE and laser-induced subretinal fibrosis and (ii) promotes or inhibits cellular senescence in the RPE. Methods: We examined laser-induced subretinal fibrosis and RPE cell contraction in wild-type and Caveolin-1 knockout (Cav-1-/-) mice treated with or without cavtratin, a cell-permeable peptide of caveolin-1. The senescence marker p16INK4a was measured in RPE tissues from patients with geographic atrophy and aged mice, laser-induced subretinal fibrosis, and primary human RPE cells. Human RPE was examined by TUNEL staining, reactive oxygen species generation, cell viability, and senescence-associated ß-galactosidase staining. Results: The volume of subretinal fibrosis was significantly smaller in cavtratin-injected eyes from wild-type mice than in control eyes from wild-type, P = 0.0062, and Cav-1-/- mice, P = 0.0095. Cavtratin treatment produced significant improvements in primary RPE cell contraction in wild-type, P = 0.04, and Cav-1-/- mice, P = 0.01. p16INK4a expression in the RPE was higher in patients with than without geographic atrophy. p16INK4a was expressed in 18-month-old but not 2-month-old wild-type mouse eyes. p16INK4a and collagen type I antibodies showed co-localization in subretinal fibrosis. Cavtratin did not affect RPE cell apoptosis or reactive oxygen species generation, but decreased cell viability and increased senescence-associated ß-galactosidase-positive cells. Conclusions: Enhanced expression of caveolin-1 successfully blocked epithelial-mesenchymal transition of RPE and the reduction of subretinal fibrosis in mice. Nevertheless, in exchange for blocking subretinal fibrosis, caveolin-1 promotes RPE cellular senescence and might affect the progression of geographic atrophy in AMD.

Caveolin-1 scaffolding domain peptide regulates glucose metabolism in lung fibrosis.

Increased metabolism distinguishes myofibroblasts or fibrotic lung fibroblasts (fLfs) from the normal lung fibroblasts (nLfs). The mechanism of metabolic activation in fLfs has not been fully elucidated. Furthermore, the antifibrogenic effects of caveolin-1 scaffolding domain peptide CSP/CSP7 involving metabolic reprogramming in fLfs are unclear. We therefore analyzed lactate and succinate levels, as well as the expression of glycolytic enzymes and hypoxia inducible factor-1α (HIF-1α). Lactate and succinate levels, as well as the basal expression of glycolytic enzymes and HIF-1α, were increased in fLfs. These changes were reversed following restoration of p53 or its transcriptional target microRNA-34a (miR-34a) expression in fLfs. Conversely, inhibition of basal p53 or miR-34a increased glucose metabolism, glycolytic enzymes, and HIF-1α in nLfs. Treatment of fLfs or mice having bleomycin- or Ad-TGF-ß1-induced lung fibrosis with CSP/CSP7 reduced the expression of glycolytic enzymes and HIF-1α. Furthermore, inhibition of p53 or miR-34a abrogated CSP/CSP7-mediated restoration of glycolytic flux in fLfs in vitro and in mice with pulmonary fibrosis and lacking p53 or miR-34a expression in fibroblasts in vivo. Our data indicate that dysregulation of glucose metabolism in fLfs is causally linked to loss of basal expression of p53 and miR-34a. Treatment with CSP/CSP7 constrains aberrant glucose metabolism through restoration of p53 and miR-34a.

Involvement of caveolin-1 in neurovascular unit remodeling after stroke: Effects on neovascularization and astrogliosis.

Complex cellular and molecular events occur in the neurovascular unit after stroke, such as blood-brain barrier (BBB) dysfunction and inflammation that contribute to neuronal death, neurological deterioration and mortality. Caveolin-1 (Cav-1) has distinct physiological functions such as caveolae formation associated with endocytosis and transcytosis as well as in signaling pathways. Cav-1 has been proposed to be involved in BBB dysfunction after brain injury; however, its precise role is poorly understood. The goal of this study was to characterize the expression and effect of Cav-1 deletion on outcome in the first week in a transient Middle Cerebral Artery Occlusion stroke model. We found increased Cav-1 expression in new blood vessels in the lesion and in reactive astrocytes in the peri-lesion areas. In Cav-1 KO mice, the lesion volume was larger and the behavioral outcome worse than in WT mice. Cav-1 KO mice exhibited reduced neovascularization and modified astrogliosis, without formation of a proper glial scar around the lesion at three days post injury, coinciding with aggravated outcomes. Altogether, these results point towards a potential protective role of endogenous Cav-1 in the first days after ischemia by promoting neovascularization, astrogliosis and scar formation.

NOS3 Deletion in Cav1 Deficient Mice Decreases Drug Sensitivity to a Nitric Oxide Donor and Two Nitric Oxide Synthase Inhibitors.

Purpose: This study aims to investigate the pharmacologic consequence of genetic deletion of nitric oxide synthase 3 (NOS3) in caveolin 1 (Cav1)-/- mice (double knockout [DKO]) in response to a nitric oxide (NO) donor and two NOS inhibitors. Methods: NO donor sodium nitroprusside (SNP; 10-40 mg/mL), NOS inhibitor L-NG-nitroarginine methyl ester (L-NAME; 10-200 µM), and cavtratin (10-75 µM ) was administered topically to the eye while the contralateral eyes were vehicle controls. Intraocular pressure (IOP) was measured in both eyes by tonometry. Cyclic guanosine monophosphate (cGMP) level in outflow tissue was measured by ELISA assay. Protein expression were analyzed by western blot. Results: Inducible NOS (iNOS) expression significantly increased in the DKO mice compared with the wild type (WT), Cav1 knockout (Cav1 KO), and NOS3 KO mice. In contrast to WT, Cav1 KO and NOS3 KO mice, SNP concentration of up to 30 mg/mL did not significantly affect IOP in DKO mice. However, higher concentration (40 mg/mL) SNP significantly reduced IOP by 14% (n = 8, P < 0.01). Similarly, only 200 µM L-NAME produced a significant increase in IOP (n = 10, P < 0.05). Cavtratin did not significantly change IOP in DKO and NOS3 KO mice. cGMP activity in DKO mice was significantly lower than Cav1 KO mice (n = 4, P < 0.05). Conclusions: In conclusion, our results demonstrated that genetic deletion of NOS3 in Cav1 deficient mice resulted in reduced sensitivity to the NO donor SNP and the two NOS inhibitors possibly due to compromised NOS and cGMP activity.