Use and relevance of a bovine mammary gland explant model to study infection responses in bovine mammary tissue

Our aim was to develop an explant model to define more precisely the early response of bovine mammary epithelial cells to infection. Therefore we investigated the mRNA expression encoding for some soluble immunological factors in lipopolysaccharide (LPS)-treated bovine mammary gland explants. Explants were taken out from the mammary gland of eight lactating cows after slaughter then incubated with LPS (10 mug/ml) for 6 h. The mRNA expression of alpha-lactalbumin (alpha-la), various cytokines, tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-8, and two immunoglobulin receptors, the neonatal Fc receptor (FcRn) and polymeric immunoglobulin receptor (pIGR), were assessed with qPCR before and after 3 h and 6 h of LPS challenge. Both immunoglobulin receptors and alpha-la increased at 3 h then recovered their initial level at 6 h whereas IL-1beta, IL-6 and IL-8 increased only after 6 h (P<0.05). Surprisingly, TNF-alpha transcripts did not show any regulation in response to the LPS treatment. We nevertheless concluded that our model was valid to examine the short-term response of mammary epithelial cell challenged with LPS.

Lactoferrin (Lf): Retinoid interactions in the mammary glands of transgenic mice overexpressing human Lf

Induction of protein expression in a tissue-specific manner by gene transfer over-expression techniques has been one means to define the function of a protein in a biological paradigm. Studies with retinoid reporter constructs transfected in mammary cell lines suggests that lactoferrin (Lf) affects retinoid signaling pathways and alters apoptosis. We tested the effects and interactions of over-expressed mammary-specific human lactoferrin (hLf) and dietary retinol palmitate on lactation and mammary gland development in mice. Increased retinol palmitate in the diet increased daily retinol equivalents (RE) to 2.6-fold over the normal mouse control diet. Transgene (Tg) expression in the dam fed control diet depressed pup weight gain. Severe depression of pup weight gain was observed when homozygote TgTg dams were fed the RE diet. Normal weight gain was restored when pups were placed with a wild type dam fed the RE diet; conversely, normal growing pups from the wild type dams showed declining weight gains when fostered to the TgTg RE-fed dams. Northern analysis of mammary tissue extracts showed a reduction in WAP and an increase in IGFBP-3 mRNA that was associated with the presence of the transgene. Histological evaluation of 3 days lactating mammary tissue showed mammary epithelial cells from TgTg animals contained excessive secretory products, suggesting a block in cellular secretion mechanisms. In addition, the mammary cells displayed a cellular apical membrane puckering that extended into the alveoli lumens. These studies demonstrate an in vivo interaction of Tg-hLf expression and dietary retinoids in mouse mammary glands. While normal mammary gland physiology may not be representative by these experiments because high Lf concentrations during early lactation are abnormal, the demonstrated biological interaction suggests that typical periods of high Lf concentrations may have impact upon developing and involuting mammary glands.

Different vascular smooth muscle cell apoptosis in the human internal mammary artery and the saphenous vein. Implications for bypass graft disease

BACKGROUND: The remarkable patency of internal mammary artery (MA) grafts compared to saphenous vein (SV) grafts has been related to different biological properties of the two blood vessels. We examined whether proliferation and apoptosis of vascular smooth muscle cells (VSMC) from human coronary artery bypass vessels differ according to patency rates. METHODS AND RESULTS: Proliferation rates to serum or platelet-derived growth factor (PDGF)-BB were lower in VSMC from MA than SV. Surface expression of PDGF beta-receptor was slightly lower, while that of alpha-receptor was slightly higher in MA than SV. Cell cycle distribution, expression of cyclin E, cdk2, p21, p27, p57, and cdk2 kinase activity were identical in PDGF-BB-stimulated cells from MA and SV. However, apoptosis rates were higher in MA than SV determined by lactate dehydrogenase release, DNA fragmentation, and Hoechst 33258 staining. Moreover, caspase inhibitors (Z-VAD-fmk, Boc-D-fmk) abrogated the different proliferation rates of VSMC from MA versus SV. Western blotting and GSK3-beta kinase assay revealed lower Akt activity in VSMC from MA versus SV, while total Akt expression was identical. Adenoviral transduction of a constitutively active Akt mutant abrogated the different proliferation rates of VSMC from MA versus SV. CONCLUSIONS: Higher apoptosis rates due to lower Akt activity rather than different cell cycle regulation account for the lower proliferation of VSMC from MA as compared to SV. VSMC apoptosis may protect MA from bypass graft disease.

10-year follow-up of a prospective randomized trial comparing bare-metal stenting with internal mammary artery grafting for proximal, isolated de novo left anterior coronary artery stenosis the SIMA (Stenting versus Internal Mammary Artery grafting) trial

OBJECTIVES: This study was designed to compare the long-term clinical outcome of coronary artery bypass grafting (CABG) with intracoronary stenting of patients with isolated proximal left anterior descending coronary artery. BACKGROUND: Although numerous trials have compared coronary angioplasty with bypass surgery, none assessed the clinical evaluation in the long term. METHODS: We evaluated the 10-year clinical outcome in the SIMA (Stent versus Internal Mammary Artery grafting) trial. Patients were randomly assigned to stent implantation versus CABG. RESULTS: Of 123 randomized patients, 59 underwent CABG and 62 received a stent (2 patients were excluded). Follow-up after 10 years was obtained for 98% of the randomized patients. Twenty-six patients (42%) in the percutaneous coronary intervention group and 10 patients (17%) in the CABG group reached an end point (p < 0.001). This difference was due to a higher need for additional revascularization. The incidences of death and myocardial infarction were identical at 10%. Progression of the disease requiring additional revascularization was rare (5%) and was similar for the 2 groups. Stent thrombosis occurred in 2 patients (3%). Angina functional class showed no significant differences between the 2 groups. CONCLUSIONS: Both stent implantation and CABG are safe and highly effective in relieving symptoms in patients with isolated, proximal left anterior descending coronary artery stenosis. Stenting with bare-metal stents is associated with a higher need for repeat interventions. The long-term prognosis for these patients is excellent with either mode of revascularization.

Differential expression of ABC transporters and their regulatory genes during lactation and dry period in bovine mammary tissue

ATP-binding cassette (ABC) transporters play a pivotal role in human physiology, and mutations in these genes often result in severe hereditary diseases. ABC transporters are expressed in the bovine mammary gland but their physiological role in this organ remains elusive. Based on findings in the context of human disorders we speculated that candidate ABC transporters are implicated in lipid and cholesterol transport in the mammary gland. Therefore we investigated the expression pattern of selected genes that are associated with sterol transport in lactating and nonlactating mammary glands of dairy cows. mRNA levels from mammary gland biopsies taken during lactation and in the first and second week of the dry period were analysed using quantitative PCR. Five ABC transporter genes, namely ABCA1, ABCA7, ABCG1, ABCG2 and ABCG5, their regulating genes LXRalpha, PPARgamma, SREBP1 and the milk proteins lactoferrin and alpha-lactalbumin were assessed. A significantly enhanced expression in the dry period was observed for ABCA1 while a significant decrease of expression in this period was detected for ABCA7, ABCG2, SREBP1 and alpha-lactalbumin. ABCG1, ABCG5, LXRalpha, PPARgamma and lactoferrin expression was not altered between lactation and dry period. These results indicate that candidate ABC transporters involved in lipid and cholesterol transport show differential mRNA expression between lactation and the dry period. This may be due to physiological changes in the mammary gland such as immigration of macrophages or the accumulation of fat due to the loss of liquid in the involuting mammary gland. The current mRNA expression analysis of transporters in the mammary gland is the prerequisite for elucidating novel molecular mechanisms underlying cholesterol and lipid transfer into milk.

SFRP-4 abrogates Wnt-3a-induced beta-catenin and Akt/PKB signalling and reverses a Wnt-3a-imposed inhibition of in vitro mammary differentiation

ABSTRACT: BACKGROUND: Conserved Wnt ligands are critical for signalling during development; however, various factors modulate their activity. Among these factors are the Secreted Frizzled-Related Proteins (SFRP). We previously isolated the SFRP-4 gene from an involuting rat mammary gland and later showed that transgenic mice inappropriately expressing SFRP-4 during lactation exhibited a high level of apoptosis with reduced survival of progeny. RESULTS: In order to address the questions related to the mechanism of Wnt signalling and its inhibition by SFRP-4 which we report here, we employed partially-purified Wnt-3a in a co-culture model system. Ectopic expression of SFRP-4 was accomplished by infection with a pBabepuro construct. The co-cultures comprised Line 31E mouse mammary secretory epithelial cells and Line 30F, undifferentiated, fibroblast-like mouse mammary cells. In vitro differentiation of such co-cultures can be demonstrated by induction of the beta-casein gene in response to lactogenic hormones.We show here that treatment of cells with partially-purified Wnt-3a initiates Dvl-3, Akt/PKB and GSK-3beta hyperphosphorylation and beta-catenin activation. Furthermore, while up-regulating the cyclin D1 and connexin-43 genes and elevating transepithelial resistance of Line 31E cell monolayers, Wnt-3a treatment abrogates differentiation of co-cultures in response to the lactogenic hormones prolactin, insulin and glucocorticoid. Cells which express SFRP-4, however, are largely unaffected by Wnt-3a stimulation. Since a physical association between Wnt-3a and SFRP-4 could be demonstrated with immunoprecipitation/Western blotting experiments, this interaction, presumably owing to the Frizzled homology region typical of all SFRPs, explains the refractory response to Wnt-3a which was observed. CONCLUSION: This study demonstrates that Wnt-3a treatment activates the Wnt signalling pathway and interferes with in vitro differentiation of mammary co-cultures to beta-casein production in response to lactogenic hormones. Similarly, in another measure of differentiation, following Wnt-3a treatment mammary epithelial cells could be shown to up-regulate the cyclin D1 and connexin-43 genes while phenotypically they show increased transepithelial resistance across the cell monolayer. All these behavioural changes can be blocked in mammary epithelial cells expressing SFRP-4. Thus, our data illustrate in an in vitro model a mechanism by which SFRP-4 can modulate a differentiation response to Wnt-3a.

The Shelhigh No-React bovine internal mammary artery: a questionable alternative conduit in coronary bypass surgery?

BACKGROUND: Increasing age and comorbidities among patients undergoing coronary artery bypass surgery (CABG) stimulates the exhaustive research for alternative grafts. No-React treatment should render the tissue resistant against degeneration and reduce early inflammatory response. The aim of the present study was an invasive assessment of the patency of No-React bovine internal mammary artery (NRIMA grafts) used as bypass conduit in CABG surgery. PATIENTS AND METHODS: Nineteen NRIMA grafts were used in 17 patients (2.9%) out of a total of 572 patients undergoing CABG surgery within a 12-month period. All intraoperative data were assessed and in-hospital outcome was analysed. Follow-up examination was performed 7.0+/-4.0 months after initial surgery, including clinical status and coronary angiography to assess patency of the NRIMA grafts. RESULTS: Average perioperative flow of all NRIMA grafts was 71+/-60 ml/min. One patient died in hospital due to a multi-organ failure. Four patients refused invasive assessment. Follow-up was complete in 12 patients with overall 13 NRIMA grafts. Nine NRIMA grafts (69.2%) were used for the right coronary system, two NRIMA grafts (15.4%) on the LAD and two on the circumflex artery. Graft patency was 23.1% and was independent of the intraoperative flow measurement. CONCLUSIONS: NRIMA grafts show a very low patency and cannot be recommended as coronary bypass graft conduits. Patency was independent of the perioperative flow, assessed by Doppler ultrasound. Because of this unsatisfying observation, this type of graft should be utilised as a last resource conduit and used only to revascularise less important target vessels, such as the end branches of the right coronary artery.

Proliferation and apoptosis in mammary epithelium during the rat oestrous cycle

AIM: During each oestrous cycle, the mammary gland is subject to changes in ovarian hormone levels. It responds with limited proliferation, differentiation and regression. To understand the processes resulting in these changes, particularly the regulation of cell death, we examined protein levels in mammary epithelium during the oestrous cycle of the Sprague-Dawley rat. METHODS: Studies of serum hormone levels, vaginal smears, uterine weight and morphology, mammary gland morphology, proliferation and apoptotic indices, and protein levels during the stages of the Sprague-Dawley rat oestrous cycle were used to examine the response of mammary epithelium to the oestrous cycle. RESULTS: Proliferation of mammary epithelium was greater in diestrus and proestrus, while apoptosis was increased in metestrus and diestrus. Growth factor-, hormone- and anchorage-mediated cell survival signalling, indicated by activation of Stat5A, FAK and Akt 1 and expression of anti-apoptotic Bcl-2 family members, was greater in proestrus and reduced in metestrus. In contrast, the levels of pro-apoptotic Bcl-2 family members and proteins associated with apoptosis in mammary epithelium (TGFbeta3, pStat3) were increased during metestrus and diestrus. CONCLUSION: Decreases in growth factor, hormone and cell attachment survival signals corresponded with increased apoptosis during the second half of the oestrous cycle. The protein levels detected during oestrus suggest parallels to apoptosis in mammary involution.

Non-robotic thoracoscopic internal mammary artery preparation in the pig. A training model

Notwithstanding non-robotic, thoracoscopic preparation of the internal mammary artery (IMA) is a difficult surgical task, an appropriate experimental training model is lacking. We evaluated the young domestic pig for this purpose. Four domestic female pigs (30-40 kg body weight) were used for this study. Bilateral thoracoscopic preparation of the IMA was carried out under continuous, pressure controlled CO(2) insufflation. A 30 degrees rigid thoracoscope was inserted through a 10-mm port in the 5th/6th intercostal space (ICS) dorsally to the posterior axillary line. The dissection instrument (Ultracision Harmonic Scalpel) was inserted (5-mm port) in the 7th ICS at the posterior axillary line and the endo-forceps (5-mm port) in the 5th ICS at the posterior axillary line. Thoracoscopic IMA preparation in pig resulted more difficult than in man. A total of seven IMAs were prepared in their full intrathoracic length. A change in the preparation technique (lateral detachment of the endothoracic muscle) improved the safety of the procedure, allowing all four respective IMAs to be prepared safely, while the initial technique ensued an injury for 2 out of 3 vessels. The described young domestic pig model is suitable for experimental training of bilateral thoracoscopic IMA preparation.

Apoptotic cell death in the lactating mammary gland is enhanced by a folding variant of alpha-lactalbumin

Apoptosis is essential to eliminate secretory epithelial cells during the involution of the mammary gland. The environmental regulation of this process is however, poorly understood. This study tested the effect of HAMLET (human alpha-lactalbumin made lethal to tumor cells) on mammary cells. Plastic pellets containing HAMLET were implanted into the fourth inguinal mammary gland of lactating mice for 3 days. Exposure of mammary tissue to HAMLET resulted in morphological changes typical for apoptosis and in a stimulation of caspase-3 activity in alveolar epithelial cells near the HAMLET pellets but not more distant to the pellet or in contralateral glands. The effect was specific for HAMLET and no effects were observed when mammary glands were exposed to native a-lactalbumin or fatty acid alone. HAMLET also induced cell death in vitro in a mouse mammary epithelial cell line. The results suggest that HAMLET can mediate apoptotic cell death in mammary gland tissue.