Lactic acid induces HSPA1A expression through ERK1/2 activation.

Heat shock protein (HSP) A1A protects cells from various stressors. The concentrated liquid of the traditional Japanese rice black vinegar Kurozu increased HSPA1A expression in normal rat liver RLN-10 cells. Lactic acid, the primary component of concentrated Kurozu, induced HSPA1A expression in a concentration-dependent manner. Induction with 4 m m lactic acid increased HSPA1A expression by three times compared with that in the absence of lactic acid. The induction was inhibited by staurosporine or a selective MEK1/2 inhibitor (SL327). The phosphorylation of ERK1/2 was increased by lactic acid. These results suggest that lactic acid induces HSPA1A expression by activating ERK1/2. As well as lactate, 3,5-dihydroxybenzoic acid (DHBA), a ligand for G protein-coupled receptor 81 (GPR81), also induced HSPA1A at lower concentrations than lactate. The increased effect of DHBA on HSPA1A expression as compared with lactate may be related to the higher affinity of DHBA for GPR81 than of lactate.

Association of blood pressure and dietary intake of Sunomono, Japanese vinegared side dishes, in community-dwelling Japanese: A cross-sectional study.

Objective: Vinegar has been reported to have a hypotensive effect. We aimed to investigate the relationship between the consumption of vinegar-based side dishes and blood pressure. Research methods & procedures: This cross-sectional study included 746 individuals (257 men and 489 women) aged ≥40 years from Tarumizu, Kagoshima, Japan. Nutrient intake was estimated based on the brief-type self-administered diet history questionnaire. The intake frequency of vinegar-based side dishes (Sunomono and pickles) was determined using a self-administered diet history questionnaire. Participants who did not consume vinegar-based side dishes for a month were defined as having no Sunomono or pickle eating habit. Blood pressure was categorized into four groups according to the Japanese Society of Hypertension Guidelines for the Management of Hypertension. The association between the intake of vinegar-based side dishes and blood pressure categories was analyzed using ordinal logistic regression analysis adjusted for age, body mass index, smoking history, excessive alcohol intake, living situation, energy intake, protein intake, sodium intake, potassium intake, and seaweed intake. Results: Approximately 13.6% men and 6.1% women had no Sunomono eating habits. In men, eating Sunomono, but not pickles, was significantly related to blood pressure categories (estimate, -0.702; 95% CI, -1.122 to -0.310), whereas more frequent consumption of Sunomono did not show an improvement in the blood pressure category. The relationship between eating Sunomono and blood pressure categories was not recognized in women. Conclusion: This was the first study assessing the association between consumption of vinegar-based side dishes and blood pressure categories. We highlighted the effect of Sunomono consumption on blood pressure categories in men. Consumption of Sunomono may improve blood pressure in men.

Relationship between Oral Hypofunction, and Protein Intake: A Cross-Sectional Study in Local Community-Dwelling Adults.

Few studies have investigated the relationship between nutritional status and comprehensive assessment of oral hypofunction, especially protein intake-related sarcopenia. Thus, we explored these relationships in a large-scale cross-sectional cohort study using the seven-item evaluation for oral hypofunction and Diet History Questionnaire for nutritional assessment. We used the data from 1004 individuals who participated in the 2019 health survey of the residents of Tarumizu City, Kagoshima Prefecture, Japan for analysis. We found that individuals with oral hypofunction were significantly older with a lower skeletal muscle index. Although there were few foods that had a significant difference between the groups with and without oral hypofunction, the consumption of beans and meats was significantly lower in women and men in the oral hypofunction group, respectively. According to the lower limit of the tentative dietary goal defined in Japan, comprehensive evaluation of oral hypofunction was significantly and independently associated with protein intake in both men and women (odds ratio, 1.70; 95% confidence interval, 1.21-2.35). In conclusion, we found that oral hypofunction was associated with targeted protein intake for sarcopenia and frailty prevention in middle-aged and older community-dwelling adults. Comprehensive evaluation of oral function with intervention in cases of hypofunction could inform clinicians to better prevent sarcopenia.

Association of Protein and Magnesium Intake with Prevalence of Prefrailty and Frailty in Community-Dwelling Older Japanese Women.

We examined the association between nutrient intake and prefrailty. Data from 815 older people (63% women) who participated in a community-based health check survey (Tarumizu Study) were analyzed. Prefrailty were defined using five parameters (exhaustion, slowness, weakness, low physical activity, and weight loss). Participants with one or more components were considered to belong to the prefrailty group. Nutrition intake was estimated from a validated brief-type self-administered diet history questionnaire. Among the participants, 154 men (52%) and 278 women (54%) were found to be in a status of prefrailty. In men, there were no significant associations between nutrient intake and prefrailty. In women, carbohydrate intake was slightly higher in prefrailty group. Vitamins K, B1, B2, folic acid, pantothenic acid, phosphorus, potassium, calcium, magnesium, iron, zinc, and copper intake was significantly lower in the prefrailty group. Among the nutrients, magnesium was identified as a significant covariate of prefrailty using a stepwise regression method. In women adjusted ORs (95%CI, p value) for prefrailty in the first, second, third, and fourth quartiles of magnesium intake were 1.00 (reference), 0.52 (0.29-0.92, 0.024), 0.51 (0.28-0.95, 0.033), and 0.38 (0.19-0.74, 0.005), respectively, by multivariate logistic regression analysis (variates: age, body mass index, energy intake, supplement use, osteoporosis, magnesium, and protein intake). Protein intake did not related to prefrailty. Protein intake might be sufficient to prevent prefrailty in the present study. We propose magnesium to be an important micronutrient that prevents prefrailty in community-dwelling older Japanese women.

The Association between Dietary Variety and Physical Frailty in Community-Dwelling Older Adults.

The aim of this cross-sectional study was to examine the association between diet variety and physical frailty in community-dwelling older adults. Data of 577 older adults (mean age: 74.0 ± 6.3 years, women: 62.5%) were analyzed. Diet variety was assessed using the Food Frequency Score (FFS) (maximum, 30 points). The FFS assessed the one-week consumption frequency of ten foods (meat, fish/shellfish, eggs, milk & dairy products, soybean products, green & yellow vegetables, potatoes, fruits, seafood, and fats & oil). Physical frailty was assessed using Fried's component (slowness, weakness, exhaustion, low physical activity, and weight loss). The participants were classified into frail, pre-frail, and non-frail groups. The prevalence of physical frailty was 6.6%. This study found significant associations between physical frailty and low FFS after adjusting for covariates (odds ratio (OR) 0.90, 95% confidence interval (CI) 0.84-0.97, p < 0.01). The optimal cutoff point of the FFS for physical frailty was ≤16 points. FFS lower than the cutoff point were significantly associated with physical frailty after adjusting for covariates (OR 3.46, 95% CI 1.60-7.50, p < 0.01). Diet variety assessed using the FFS cutoff value of ≤16 points was related to the physical frailty status in community-dwelling older adults.

The Induction of Heat Shock Protein 70 after Oral Administration of Concentrated Brewed Rice Vinegar Kurozu in Mice.

Heat shock protein 70 (HSP70) is induced by various stresses. Since HSP70 has a protein refolding activity and an anti-inflammatory activity, the HSP70 induction will help cells from harmful acute stresses. Feeding a diet containing concentrated brewed rice vinegar Kurozu (CK) diet for 5 wk resulted in an increase of HSP70 in the brains of mice. In the present study, we evaluated whether oral feeding of 25 µL CK induces HSP70 mRNA in brain and other tissues. HSP70 mRNA was significantly increased in the esophagus, small intestine, liver, and brown adipose tissue within 1 h after the oral administration of CK. A weaker induction of HSP70 mRNA was demonstrated in the stomach, large intestine, and brain. HSP70 mRNA induction returned to basal levels within 3 h after feeding. We doubted that the induction of HSP70 mRNA was caused by manual restraint of the mice during CK administration. Manual restraint of the mice did not influence HSP70 mRNA expression in intestine 1 h after these treatments. Our results suggest that transient HSP70 mRNA induction by oral feeding of CK was not caused by retention stress. There are some compounds in CK that increase HSP70 mRNA in various tissues.

Effect of dietary fermented mushroom bed on egg production in laying hens.

Egg productivity is declined by stress. It has been reported that some food supplements can improve the egg productivity due to a reduction of environmental stress. We evaluated the effect of fermented waste mushroom bed (FWMB) as a dietary additive on egg productivity. Hens were fed control food (control group, n = 100) or 3% FWMB-added food (FWMB group, n = 100) for 16 months. The number of eggs, soft-shelled eggs, and broken eggs were recorded for 15 months. We also evaluated stress-related markers (ovotransferrin, lipid peroxide, and the heterophil-to-lymphocyte ratio). The FWMB group had slightly increased egg production compared with control hens. The FWMB group produced significantly less broken and soft-shelled eggs than the control group. All stress-related markers were significantly lower in the FWMB group than in the control group. Gut flora was also affected by FWMB feeding. The increased egg production and decreased proportion of broken and soft-shelled eggs might be related to the prevention of stressful conditions by FWMB.

The Brewed Rice Vinegar Kurozu Increases HSPA1A Expression and Ameliorates Cognitive Dysfunction in Aged P8 Mice.

Kurozu is a traditional Japanese rice vinegar. During fermentation and aging of the Kurozu liquid in an earthenware jar over 1 year, a solid residue called Kurozu Moromi is produced. In the present study, we evaluated whether concentrated Kurozu or Kurozu Moromi could ameliorate cognitive dysfunction in the senescence-accelerated P8 mouse. Senescence-accelerated P8 mice were fed 0.25% (w/w) concentrated Kurozu or 0.5% (w/w) Kurozu Moromi for 4 or 25 weeks. Kurozu suppressed cognitive dysfunction and amyloid accumulation in the brain, while Kurozu Moromi showed a tendency to ameliorate cognitive dysfunction, but the effect was not significant. We hypothesize that concentrated Kurozu has an antioxidant effect; however, the level of lipid peroxidation in the brain did not differ in senescence-accelerated P8 mice. DNA microarray analysis indicated that concentrated Kurozu increased HSPA1A mRNA expression, a protein that prevents protein misfolding and aggregation. The increase in HSPA1A expression by Kurozu was confirmed using quantitative real-time PCR and immunoblotting methods. The suppression of amyloid accumulation by concentrated Kurozu may be associated with HSPA1A induction. However, concentrated Kurozu could not increase HSPA1A expression in mouse primary neurons, suggesting it may not directly affect neurons.

Positive Association of Plasma Homocysteine Levels with Cardio-Ankle Vascular Index in a Prospective Study of Japanese Men from the General Population.

AIM: Observational studies have reported that elevated homocysteine (Hcy) levels are associated with the risk of cardiovascular disease (CVD). However, interventions that lower Hcy do not provide a corresponding risk reduction. Therefore, the causal role of Hcy in CVD remains unclear. This 5-year prospective study investigated the associations of Hcy levels, folate intake, and host factors with arterial stiffness among the general Japanese population. METHODS: We prospectively recruited 658 participants (40-69 years old) from the general population during regular health checkup examinations. Arterial stiffness was evaluated using the cardio-ankle vascular index (CAVI) at baseline and the 5-year follow-up. Folate intake was estimated using a structured questionnaire. Genotyping was used to evaluate the MTHFR C677T and MS A2756G gene polymorphisms. Ultrafast liquid chromatography was used to measure total plasma Hcy levels. Association between these variables and CAVI values was evaluated using general linear regression and logistic regression models that were adjusted for atherosclerosis-related factors. RESULTS: Men had higher Hcy levels and CAVI values and lower folate intake than women (all, p＜0.001). At baseline, Hcy, folate intake, and the two genotypes were not associated with CAVI values for both sexes. Among men, Hcy levels were positively associated with CAVI values at the 5-year follow-up (p=0.033). Folate intake and the two genotypes were not associated with the 5-year CAVI values. CONCLUSION: Plasma Hcy may be involved in arterial stiffness progression, as monitored using CAVI, among men.

Ethanol extract of Brazilian propolis ameliorates cognitive dysfunction and suppressed protein aggregations caused by hyperhomocysteinemia.

Homocysteine (Hcy) has been proposed to be a risk factor for cognitive dysfunction. We investigated the effects and the underlying mechanisms of action of propolis, which has antioxidant activity on Hcy-induced oxidative stress in vitro and in vivo. For the in vitro assays, neuroblastoma SH-SY5Y and glioblastoma U-251MG cells were cultured with Hcy and various concentrations of propolis. Cell death and reactive oxygen species production were significantly suppressed by propolis in dose-dependent manner, compared with Hcy alone. For the in vivo assays, mice were fed a propolis-containing diet and Hcy thiolactone in water. Cognitive function was evaluated using the Morris water maze test. Propolis suppressed cognitive dysfunction caused by hyperhomocysteinemia. Accumulation of aggregated protein in brain was accelerated in hyperhomocysteinemia, and the accumulation was suppressed by propolis. Hyperhomocysteinemia, however, did not enhance the oxidative stress in brain. In vitro amyloid formation assay showed that Hcy accelerated lysozyme aggregation and propolis inhibited the aggregation.

Growth suppression and cell death by pyridoxal is dependent on p53 in the human breast cancer cell line MCF-7.

Vitamin B6 compound, pyridoxine (PN), has shown antitumor action. Our previous experiments showed that PN induces expression of insulin-like growth factor binding protein-3 to arrest proliferation and induce cell death. This induction is inhibited by the p53-specific inhibitor pifithrin-α. Here, we report that another B6 compound, pyridoxal (PL), strongly inhibited MCF-7 cell growth compared to PN. PL induced the G0/G1 arrest and the accumulation of subG1 population. Although p53 mRNA was not changed by PL, 0.5 mM PL increased the protein level in MCF-7 cells. The cell growth suppression by 0.5 mM PL did not occur when p53 expression was knocked down using siRNA. Together, these data suggest that PL accumulate p53 and PL-induced cell growth suppression is dependent on p53 in MCF-7 breast cancer cells.

Plasma homocysteine concentrations in novel microminipigs.

A novel microminipig has been recently developed for use in biomedical research. In the present study, age- and sex-related differences, as well as 24-h fluctuations in plasma total homocysteine concentrations (tHcy), were investigated in these microminipigs. tHcy (mean±SD) was 10.2±3.4 µM and significantly correlated with age. By contrast, neither the differences in tHcy between sexes nor the 24-h fluctuations in tHcy after feeding were significant. The kinetics of plasma tHcy after intravenous injection of reduced Hcy showed that its levels peaked within 5 min post-injection, as did the levels of tHcy. These results suggested that reduced Hcy is rapidly oxidized or metabolized. The half-lives of reduced Hcy, tHcy, and reduced cysteine in the blood were 47, 71, and 141 min, respectively. In conclusion, there was a significantly positive correlation between age and plasma tHcy in microminipigs. After intravenous injection of reduced Hcy, plasma tHcy quickly returned to pre-injection levels.

Low pyridoxine concentrations enhance lipopolysaccharide-stimulated gene expression of cyclooxygenase-2 and inducible nitric oxide synthase in RAW264.7 cells.

Pyridoxal (PL) has been shown to suppress lipopolysaccharide (LPS)-induced gene expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Commonly used cell culture media contain extremely high concentrations of pyridoxine (PN) compared to total serum levels of vitamin B6. Therefore, we evaluated how physiological concentrations of PN influence LPS-stimulated gene expression of COX-2 and iNOS. The mouse macrophage cell line, RAW264.7, was cultured in PN-free DMEM supplemented with 10% fetal bovine serum (DMEM(-PN+FBS)) for 7 d. Although the level of pyridoxal 5'-phosphate in these cells was decreased by 65%, no change was observed in cell proliferation rate or aspartate aminotransferase activity for 7 d. LPS-induced expression of COX-2 mRNA was compared between DMEM(+FBS) and DMEM(-PN+FBS). COX-2 expression was enhanced by 2.2 or 1.9 times with a 1 or 3 d treatment, respectively; however, no difference was observed at 7 d. PN (0.032-100 µm) added to the DMEM(-PN+FBS) and RAW264.7 cells was cultured in the medium containing each concentration of PN for 1 d. Enhancement of COX-2 and iNOS gene expression was suppressed by PN addition in a concentration-dependent manner. COX-2 and iNOS mRNA were similarly expressed in cells grown in media containing PN at 4 µm or higher. Overall, induction of COX-2 and iNOS by LPS was transiently enhanced when RAW264.7 cells were cultured in physiological PN concentrations.

Supplementation with branched-chain amino acids attenuates hepatic apoptosis in rats with chronic liver disease.

Branched-chain amino acids (BCAA) can function as pharmacologic nutrients for patients with decompensated cirrhosis. However, the effects of BCAA at the early stage of chronic liver disease are not clear. We hypothesized that early BCAA supplementation would attenuate the progression of chronic liver disease. The present study examined the effects of BCAA supplementation on the progression of chronic liver disease in rats caused by injected carbon tetrachloride (CCl4). Sprague-Dawley rats were fed with a casein diet (control group) or the same diet supplemented with BCAA (BCAA group) for 11 weeks, and all rats were repeatedly injected with CCl4. Food intake did not significantly differ between control and BCAA groups during the experimental period. Plasma alanine aminotransferase activities gradually increased during the experimental period in both groups but peaked later in the BCAA group. Liver fibrosis was more evident in the control group. Levels of connective tissue growth factor messenger RNA were significantly lower in the livers of rats in the BCAA group than in the control group. Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling assays found considerably more hepatic apoptosis in the control group. Liver cytosolic cytochrome c levels and expression of the proapoptotic Bax protein in the mitochondrial fraction were significantly lower in the BCAA group than in the control group. These results suggest that supplementation with BCAA delays the progression of chronic liver disease caused by CCl4 in rats by attenuating hepatic apoptosis.

Decomposition of insoluble and hard-to-degrade animal proteins by enzyme E77 and its potential applications.

Insoluble and hard-to-degrade animal proteins are group of troublesome proteins, such as collagen, elastin, keratin, and prion proteins that are largely generated by the meat industry and ultimately converted to industrial wastes. We analyzed the ability of the abnormal prion protein-degrading enzyme E77 to degrade insoluble and hard-to-degrade animal proteins including keratin, collagen, and elastin. The results indicate that E77 has a much higher keratinolytic activity than proteinase K and subtilisin. Maximal E77 keratinolytic activity was observed at pH 12.0 and 65 °C. E77 was also adsorbed by keratin in a pH-independent manner. E77 showed lower collagenolytic and elastinolytic specificities than proteinase K and subtilisin. Moreover, E77 treatment did not damage collagens in ovine small intestines but did almost completely remove the muscles. We consider that E77 has the potential ability for application in the processing of animal feedstuffs and sausages.

Homocysteine induced SH-SY5Y apoptosis through activation of NADPH oxidase in U251MG cells.

Epidemiological studies have indicated a correlation between homocysteinemia and dementia, including Alzheimer's disease. However, the mechanism by which homocysteine (Hcy) induces neuronal cell death remains unknown. We found that micromolar concentrations of Hcy induced neuroblastoma SH-SY5Y cell death only when co-cultured with glioblastoma U251MG cells. In this culture system, cysteine had no effect on SH-SY5Y cell death. There was an increase in TUNEL-positive cells and loss of mitochondrial membrane potential following treatment with 100 µM Hcy. Addition of conditioned medium prepared from U251MG cells in the presence of 100 µM Hcy also reduced SH-SY5Y cell viability, while this effect was prevented when using conditioned medium from U251MG cells exposed to 100 µM Hcy+apocynin, a specific NADPH oxidase inhibitor. Following exposure to 100 µM Hcy in U251MG cells, expression of Rac1, a compartment of NADPH oxidase, was translocated to the plasma membrane, and the active form of Rac1 was increased. There was no change in peroxide concentration in the medium of U251MG cells after addition of Hcy. Overall, these data suggest that Hcy stimulates Rac1 activation and NADPH oxidase, resulting in superoxide anion production that may induce SH-SY5Y cell apoptosis.

Live-cell visualization of the trans-cellular mode of monocyte transmigration across the vascular endothelium, and its relationship with endothelial PECAM-1.

In response to atherogenic stimuli, blood monocytes transmigrate across the vascular endothelium not only through endothelial cell-cell junctions (para-cellular) but also through endothelial cells themselves (trans-cellular). The molecular mechanism of the latter is mostly unknown, because it rarely happens, especially in vitro. Although many reports have recognized trans-cellular migration from snapshot images of leukocytes halfway across the endothelium at non-junctional locations, it often produces a false-positive result, because some leukocytes that initiate trans-cellular migration withdraw and return to the apical endothelial surface. Thus, analyzing the entire process is essential. In this study, complete monocyte trans-cellular migration was successfully captured for live cells, with simultaneous visualization of endothelial PECAM-1. We suggest the possible existence of both PECAM-1-related migration at peri-junctional sites and PECAM-1-unrelated migration at sites remote from junctions. This is the first report to describe the entire process of monocyte trans-cellular migration for live cells and its relationship with endothelial PECAM-1.

High dose of pyridoxine induces IGFBP-3 mRNA expression in MCF-7 cells and its induction is inhibited by the p53-specific inhibitor pifithrin-α.

It has been reported that supplementation with high-dose vitamin B(6) (B(6)) exerts antitumor effects in rodent models of cancer. However, the mechanism of these effects remains poorly understood. High-dose B(6) also suppresses cell proliferation and induces apoptosis of human breast adenocarcinoma MCF-7 cells. Based on preliminary experiments using DNA microarray analyses, we hypothesized that high-dose pyridoxine (PN) might induce IGF-binding protein-3 (IGFBP-3) expression in MCF-7 cells. In this study, we investigated IGFBP-3 induction by 3 or 10 mM PN using a quantitative real-time PCR method. We found that the induction reached a maximum of 24-fold with 10 mM PN for 72 h compared with non-treated cells. The induction of IGFBP-3 by PN was inhibited by a p53-specific inhibitor, pifithrin-α, in a dose-dependent manner, but was not affected by PD169316 (MAPK inhibitor), AS601245 (c-Jun N-terminal kinase inhibitor) or SL327 (MEK1/2 inhibitor). High-dose PN did not induce p53 mRNA expression. The IGFBP-3 induction by PN seemed to be related to p53 activation.

Monocyte trans-endothelial migration augments subsequent transmigratory activity with increased PECAM-1 and decreased VE-cadherin at endothelial junctions.

BACKGROUND: Although the importance of monocyte trans-endothelial migration in early atherogenesis is well recognized, it is unclear whether and how one transmigration event affects endothelium to facilitate subsequent ones. In this study, we tested the hypothesis that monocyte transmigration alters endothelial junctional organization to facilitate subsequent transmigration. METHODS AND RESULTS: When human monocytes were added twice at intervals of ≈30 min to IL-1beta-prestimulated human umbilical vein endothelial cells in vitro, significant augmentation of transmigration was observed at the second addition (≈1.5-fold, analyzed from a total of 231 monocytes in 3 experiments). Endothelial surface expressions of two major junctional molecules, PECAM-1 and VE-cadherin, increased and decreased respectively, in response to monocyte addition, which could facilitate subsequent transmigration. To further investigate spatiotemporal dynamics of the increasing molecule, PECAM-1, we constructed a PECAM-1-GFP expression system and found that monocyte transmigration induced local accumulation of endothelial PECAM-1 around the transmigration spot, which was followed by transmigration of subsequent monocyte around the same location. Detailed analysis revealed that within the defined region around one transmigration event, 50% of later transmigrating monocytes used the same or similar location as the previous one (10 out of 20 transmigrating monocytes in 11 experiments). CONCLUSIONS: These findings show that monocyte trans-endothelial migration alters endothelial junctional organization to a more monocyte-permeable state (increased PECAM-1 and decreased VE-cadherin), resulting in the augmented transmigratory activity at a later stage. This positive feedback mechanism is partially associated with monocyte transmigration-induced local accumulation of endothelial PECAM-1, which promotes transmigration of following monocytes at the same location.

Change in VEGF expression in mouse mammary gland during reproductive cycle.

During pregnancy, the mammary epithelium and its supporting vasculature undergo extensive growth and proliferation in preparation for lactation, which is thought to be dependent on vascular endothelial growth factor (VEGF). We investigated the expression of VEGF, using immunohistochemistry and immunoblotting, in the mouse mammary gland during the reproductive cycle. Immunohistochemical studies localized VEGF protein predominantly in the cytoplasm of the mammary epithelium and revealed it to be strongly expressed in late pregnancy and early lactation. In addition, immunoblot analysis revealed a 45-kD VEGF band, corresponding to the homodimer of the VEGF-A164 isoform, with increased expression towards the end of pregnancy but no additional increase with the onset of lactation. As the post-lactation period advanced, a dramatic decrease in VEGF was observed in the regressed mammary epithelium. The expression of VEGF suggests that mammary epithelium-derived VEGF may be involved in pregnancy-associated mammary growth and differentiation and angiogenesis, and regulate vascular permeability during lactation in an autocrine or paracrine manner.