CU06-1004 as a promising strategy to improve anti-cancer drug efficacy by preventing vascular leaky syndrome.

Background: Interleukin-2 (IL-2) is the first cancer therapeutic agent with an immunomodulatory function. Although it has been experimentally proven to be effective against metastatic renal cell carcinoma and metastatic melanoma, the clinical application of high-dose IL-2 (HDIL-2) has been limited because of its short half-life and severe side effects, such as vascular leakage syndrome (VLS) or capillary leaky syndrome (CLS). However, methods for overcoming this issue have not yet been identified. Methods: We discovered CU06-1004, an endothelial dysfunction blocker, through a previous study, and co-treated with IL-2 immunotherapy to confirm its inhibitory effect on HDIL-2-induced endothelial permeability. CU06-1004 was co-administered with HDIL-2 for 4 days in an in vivo mouse model. After drug injection, the mice were sacrificed, and Evans blue staining was performed. Results: In vitro, HDIL-2 treatment decreased HUVEC stability, which was rescued by co-treatment with CU06-1004. In our mouse model, co-administration of CU06-1004 and HDIL-2 prevented HDIL-2-induced vascular leakage by normalizing endothelial cells. Notably, the HDIL-2 and CU06-1004 combination therapy considerably reduced tumor growth in the B16F10 melanoma mouse model. Conclusion: Our data suggest that CU06-1004 acts as a potential anticancer drug candidate, not only by preventing HDIL-2-induced VLS but also by enhancing the anticancer effects of HDIL-2 immunotherapy.

Efficacy of CU06-1004 via regulation of inflammation and endothelial permeability in LPS-induced acute lung injury.

BACKGROUND: Acute lung injury (ALI) is a life-threatening condition that fundamentally results from inflammation and edema in the lung. There are no effective treatments available for clinical use. Previously, we found that as a leakage blocker CU06-1004 prevents endothelial barrier disruption and enhances endothelial cell survival under inflammatory conditions. In this study, we aimed to elucidate the effect of CU06-1004 in terms of prevention of inflammation and endothelial dysfunction in an ALI mouse model. METHODS: An ALI model was established that included intraperitoneal administration of LPS. Following LPS administration, survival rates and lung wet/dry ratios were assessed. Histological analysis was performed using hematoxylin and eosin staining. Scanning electron microscopy was used to examine alveolar and capillary morphology. Cytokines such as IL-1ß, IL-6, and TNF-α were analyzed using an ELISA assay of bronchoalveolar lavage fluid (BALF) and serum. Neutrophil infiltration was observed in BALF using Wright-Giemsa staining, and myeloperoxidase (MPO) activity was assessed. Pulmonary vascular leakage was confirmed using Evans-blue dye, and the expression of junctional proteins was evaluated using immunofluorescent staining. Expression of adhesion molecules was observed using immunofluorescence staining. NF-κB activation was determined using immunohistochemistry and western blot analysis. RESULTS: Survival rates and pulmonary edema were ameliorated with CU06-1004 treatment. Administration of CU06-1004 normalized histopathological changes induced by LPS, and alveolar-capillary wall thickening was reduced. Compared with the LPS-challenged group, after CU06-1004 treatment, the infiltration of immune cells was decreased in the BALF, and MPO activity in lung tissue was reduced. Similarly, in the CU06-1004 treatment group, pro-inflammatory cytokines were significantly inhibited in both BALF and serum. Evans-blue leakage was reduced, and the expression of junctional proteins was recovered in the CU06-1004 group. Adhesion molecules were downregulated and NF-κB activation was inhibited after CU06-1004 treatment. CONCLUSIONS: These results suggested that CU06-1004 had a therapeutic effect against LPS-induced ALI via alleviation of the inflammatory response and protection of vascular integrity.

CU06-1004 alleviates vascular hyperpermeability in a murine model of hereditary angioedema by protecting the endothelium.

BACKGROUND: Over-release of the vasoactive peptide bradykinin (BK) due to mutation in the SERPING1 gene is the leading cause of hereditary angioedema (HAE). BK directly activates endothelial cells and increases vascular permeability by disrupting the endothelial barrier, leading to angioedema affecting face, lips, extremities, gastrointestinal tract, and larynx. Although various pharmacological treatment options for HAE became available during the last decade, they are presently limited and pose a major economic burden on patients. To identify additional therapeutic options for HAE, we evaluated the effect of CU06-1004, an endothelial dysfunction blocker, on BK-induced vascular hyperpermeability and the HAE murine model. METHODS: To investigate the effect of CU06-1004 on BK-induced vascular hyperpermeability in vivo, we pre-administrated WT mice with the drug and then induced vascular leakage through intravenous injection of BK and observed vascular alternation. Then, SERPING1 deficient mice were used for a HAE murine model. For an in vitro model, the HUVEC monolayer was pre-treated with CU06-1004 and then stimulated with BK. RESULTS: Bradykinin disrupted the endothelial barrier and formed interendothelial cell gaps, leading to hyperpermeability in vivo and in vitro. However, CU06-1004 treatment protected the endothelial barrier by suppressing Src and myosin light chain activation via BK and alleviated hyperpermeability. CONCLUSION: Our study shows that CU06-1004 oral administration significantly reduced vascular hyperpermeability in the HAE murine model by protecting the endothelial barrier function against BK stimulation. Therefore, protecting endothelium against BK with CU06-1004 could serve as a potential prophylactic/therapeutic approach for HAE patients.

Oral administration of CU06-1004 attenuates vascular permeability and stabilizes neovascularization in retinal vascular diseases.

Retinal vascular diseases are the leading cause of blindness worldwide. These diseases have common disease mechanisms including vascular endothelial growth factor (VEGF) signaling, hypoxia, and inflammation. Treatment of these diseases with laser therapy, anti-VEGF injections and/or steroids has significantly improved clinical outcomes. However, these strategies do not address the underlying cause of the pathology and may have harmful side effects. Pathological processes that damage retinal vessels result in vascular occlusion and impairment of the barrier properties of retinal endothelial cells, leading to excessive vascular leakage. Therefore, a new therapeutic approach is needed for the treatment of retinal vascular disease. We were able to confirm that oral administration of CU06-1004, an endothelial dysfunction blocker, inhibited retinal vascular leakage induced by vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang2). Interestingly, oral administration of CU06-1004 prevented excessive vascular leakage in the diabetic retinopathy model. In addition, CU06-1004 inhibited angiogenesis and confirmed vascular stabilization in the oxygen-induced retinopathy model and laser-induced CNV model. Taken together, CU06-1004 could be a potential therapeutic agent for the treatment of retinal vascular diseases.

CU06-1004 Alleviates Experimental Colitis by Modulating Colonic Vessel Dysfunction.

Inflammatory bowel disease is an autoimmune disease that causes chronic inflammation of the gastrointestinal tract. Endothelial dysfunction, defined by a reduced endothelial barrier and an increase in the expression of adhesion molecules, is part of the pathology of inflammatory bowel disease. In this study, we assessed the therapeutic effect of CU06-1004, an endothelial dysfunction blocker that reduces vascular hyperpermeability and inflammation in a mouse model of colitis. Acute colitis was induced in mice using 3% (w/v) dextran sodium sulfate added to their drinking water for 7 days. Twenty-four hours after the addition of dextran sodium sulfate, either mesalazine or CU06-1004 was administered orally each day. Administration of CU06-1004 significantly reduced the clinical manifestations (weight loss, diarrhea, and bloody stool) and histological changes (epithelium loss, inflammatory cell infiltration, and crypt destruction) induced by dextran sodium sulfate. Proinflammatory cytokines were also reduced, indicating that inflammation was ameliorated. From a vascular perspective, CU06-1004 reduced interrupted and tortuous vessels, enhanced junction protein expression, and reduced inflammatory adhesion molecules, indicating a broad improvement of endothelial dysfunction. Endothelial protection induced epithelial barrier restoration and decreased epithelial inflammation. Blocking endothelial dysfunction with CU06-1004 significantly ameliorated the progression of inflammatory bowel disease. Therefore, CU06-1004 may represent a potential therapeutic agent for the treatment of inflammatory bowel disease as well as other inflammatory diseases.

CLEC14A deficiency exacerbates neuronal loss by increasing blood-brain barrier permeability and inflammation.

BACKGROUND: Ischemic stroke is a main cause of mortality. Blood-brain barrier (BBB) breakdown appears to play a critical role in inflammation in patients with ischemic stroke and acceleration of brain injury. The BBB has a protective function and is composed of endothelial cells, pericytes, and astrocytes. In ischemic stroke treatments, regulation of vascular endothelial growth factor (VEGF)-A and vascular endothelial growth factor receptor (VEGFR)-2 is a crucial target despite adverse effects. Our previous study found that loss of C-type lectin family 14 member A (CLEC14A) activated VEGF-A/VEGFR-2 signaling in developmental and tumoral angiogenesis. Here, we evaluate the effects of BBB impairment caused by CLEC14A deficiency in ischemia-reperfusion injury. METHODS: In vitro fluorescein isothiocyanate (FITC)-dextran permeability, transendothelial electrical resistance (TEER) assay, and immunostaining were used to evaluate endothelial integrity. BBB permeability was assessed using Evans blue dye and FITC-dextran injection in Clec14a-/- (CLEC14A-KO) mice and wild-type mice. Middle cerebral artery occlusion surgery and behavioral assessments were performed to evaluate the neurologic damage. The change of tight junctional proteins, adhesion molecules, pro-inflammatory cytokines, and microglial were confirmed by immunofluorescence staining, Western blotting, and quantitative reverse transcription polymerase chain reaction of brain samples. RESULTS: In endothelial cells, knockdown of CLEC14A increased FITC-dextran permeability and decreased transendothelial electrical resistance; the severity of this effect increased with VEGF treatment. Immunofluorescence staining revealed that tight junctional proteins were attenuated in the CLEC14A knockdown endothelial cells. Consistent with the in vitro results, CLEC14A-KO mice that were injected with Evans blue dye had cerebral vascular leakage at postnatal day 8; wild-type mice had no leakage. We used a middle cerebral artery occlusion model and found that CLEC14A-KO mice had severe infarcted brain and neurological deficits with upregulated VEGFR-2 expression. FITC-dextran leakage was present in CLEC14A-KO mice after ischemia-reperfusion, and the numbers of tight junctional molecules were significantly decreased. Loss of CLEC14A increased the pro-inflammatory response through adhesion molecule expression, and glial cells were activated. CONCLUSIONS: These results suggest that activation of VEGFR-2 in CLEC14A-KO mice aggravates ischemic stroke by exacerbating cerebral vascular leakage and increasing neuronal inflammation after ischemia-reperfusion injury.

Effect of exercise intensity on unfolded protein response in skeletal muscle of rat.

Endoplasmic reticulum (ER) stress, unfolded protein response (UPR), and mitochondrial biogenesis were assessed following varying intensities of exercise training. The animals were randomly assigned to receive either low- (LIT, n=7) or high intensity training (HIT, n=7), or were assigned to a control group (n=7). Over 5 weeks, the animals in the LIT were exercised on a treadmill with a 10° incline for 60 min at a speed of 20 m/min group, and in the HIT group at a speed of 34 m/min for 5 days a week. No statistically significant differences were found in the body weight, plasma triglyceride, and total cholesterol levels across the three groups, but fasting glucose and insulin levels were significantly lower in the exercise-trained groups. Additionally, no statistically significant differences were observed in the levels of PERK phosphorylation in skeletal muscles between the three groups. However, compared to the control and LIT groups, the level of BiP was lower in the HIT group. Compared to the control group, the levels of ATF4 in skeletal muscles and CHOP were significantly lower in the HIT group. The HIT group also showed increased PGC-1α mRNA expression in comparison with the control group. Furthermore, both of the trained groups showed higher levels of mitochondrial UCP3 than the control group. In summary, we found that a 5-week high-intensity exercise training routine resulted in increased mitochondrial biogenesis and decreased ER stress and apoptotic signaling in the skeletal muscle tissue of rats.

Effects of berberine on hippocampal neuronal damage and matrix metalloproteinase-9 activity following transient global cerebral ischemia.

Berberine, an isoquinoline alkaloid with a long history of use in Chinese medicine, has several important pharmacological effects. Several studies have revealed that berberine has neuroprotective and neuropsychiatric effects. However, there are few reports regarding the protective effect of berberine against neuronal damage following transient global cerebral ischemia. In this study, mice were subjected to 20 min of global brain ischemia and sacrificed 72 hr later. Berberine was administered for 7 days prior to ischemia and daily until sacrifice. Mice treated with berberine showed reduced matrix metalloproteinase-9 (MMP-9) activity. Berberine inhibited gelatinase activity directly in in situ zymography and reduced neuronal damage following global ischemia. Laminin expression and NeuN expression were markedly reduced in CA1 and CA2 areas after ischemia, and berberine reduced the laminin degradation and neuronal loss. In the TUNEL assay, damaged neurons were also apparent in the CA1 and CA2 areas, and berberine reduced TUNEL-positive cells. These data demonstrate that berberine, a plant alkaloid, may protect from hippocampal neuronal damage following transient global ischemia by reducing MMP-9 activity.

The synthesis of cholesterol-based cationic lipids with trimethylamine head and the effect of spacer structures on transfection efficiency.

Five cholesterol-based cationic lipids were newly synthesized based on cholest-5-en-3ß-oxyethane-N,N,N-trimethylammonium bromide (Chol-ETA) structure where the cholesterol backbone is linked to cationic head via various lengths of ether-linked carbon spacer. The transfection efficiency of these compounds was increased in order of three (Chol-PRO)