Screening of mtr-miR156a from exosomes of dairy cow blood to milk and its regulatory effect on milk protein synthesis in BMECs.

MicroRNA (miRNA) is a type of endogenous non-coding small RNA, which is abundant in living organisms. miRNAs play an important role in regulating gene expression and myriad cellular processes by binding to target messenger RNAs through complementary base pairing, and cross-species regulation mammalian cells by plant-derived xeno-miRNAs has been described. Here, we examined the miRNA species in two alfalfa (Medicago sativa, lucerne) cultivars commonly grown in Ningxia, China: cv. Zhongmu 1 and cv. Xinyan 52. Both cultivars have good salt and drought resistance. We found that the miRNA profiles were similar between the cultivars, with a slightly higher number of miRNAs present in the newer cv. Xinyan 52, which may contribute to its improved salt and drought tolerance. miRNAs were stable during drying, and some miRNAs were increased in dry versus fresh alfalfa, suggesting some miRNAs may be upregulated during drying. Alfalfa-derived miRNAs could be detected in exosomes from serum and whey collected from dairy cows, confirming the ability of the exogenous miRNAs (xeno-miRNAs) to enter the circulation and reach the mammary epithelium. In vitro studies confirmed that overexpression of mtr-miR156a could downregulate expression of Phosphatase 2 Regulatory Subunit B'gamma ( PPP2R5D) and Phosphoinositide-3-kinase Regulatory Subunit 2 (PIK3R2). Overexpression of mtr-miR156a also modulated PI3K-AKT-mTOR signaling as well as the casein content of milk produced by bovine mammary epithelial cells. Based on the known roles of PPP2R5D and PIK3R2 in regulating the PI3K-AKT-mTOR pathway as well as the effect of PI3K-AKT-mTOR on milk protein content, our findings implicate alfalfa-derived miR156a as a new cross-species regulator of milk quality in dairy cows.

Transcriptomic analysis reveals molecular insights into lactation dynamics in Jakhrana goat mammary gland.

BACKGROUND: Goat milk is gaining popularity as a superior alternative to bovine milk due to its closer resemblance to human milk. Understanding the molecular processes underlying lactation is crucial for improving milk quality and production in goats. However, the genetic mechanisms governing lactation in goats, particularly in indigenous breeds like the Jakhrana, remain largely unexplored. RESULTS: In this study, we performed a comprehensive transcriptomic analysis of Jakhrana goat mammary glands during early and late lactation stages. We isolated milk somatic cells and conducted RNA sequencing, followed by transcript quantification and mapping against the ARS1.2 Capra hircus reference assembly. Our analysis identified differentially expressed genes (DEGs) and commonly expressed genes (CEGs) across the lactation phases. Early lactation showed enrichment of genes encoding antimicrobial peptides and lubrication proteins, while late lactation exhibited heightened expression of genes encoding major milk proteins. Additionally, DEG analysis revealed upregulation of pivotal genes, such as the ABC transporter gene MRP4, implicated in modulating milk composition and quality. CONCLUSION: Our findings provide insights into the genetic mechanisms underlying lactation dynamics in the Jakhrana goat. Understanding these mechanisms could help in improving milk production and quality in goats, benefiting both the dairy industry and consumers.

Mammary fat globules as a source of mRNA to model alterations in the expression of some milk component genes during lactation in bovines.

BACKGROUND: The milk's nutritional value is determined by its constituents, including fat, protein, carbohydrates, and minerals. The mammary gland's ability to produce milk is controlled by a complex network of genes. Thereby, the fat, protein, and lactose synthesis must be boost in milk to increase milk production efficiency. This can be accomplished by fusing genetic advancements with proper management practices. Therefore, this study aimed to investigate the association between the Lipoprotein lipase (LPL), kappa casein CSN3, and Glucose transporter 1 (GLUT1) genes expression levels and such milk components as fat, protein, and lactose in different dairy breeds during different stages of lactation. METHODS: To achieve such a purpose, 94 milk samples were collected (72 samples from 36 multiparous black-white and red-white Holstein-Friesian (HF) cows and 22 milk samples from 11 Egyptian buffaloes) during the early and peak lactation stages. The milk samples were utilized for milk analysis and genes expressions analyses using non- invasive approach in obtaining milk fat globules (MFGs) as a source of Ribonucleic acid (RNA). RESULTS: LPL and CSN3 genes expressions levels were found to be significantly higher in Egyptian buffalo than Holstein-Friesian (HF) cows as well as fat and protein percentages. On the other hand, GLUT1 gene expression level was shown to be significantly higher during peak lactation than early lactation. Moreover, lactose % showed a significant difference in peak lactation phase compared to early lactation phase. Also, fat and protein percentages were significantly higher in early lactation period than peak lactation period but lactose% showed the opposite pattern of Egyptian buffalo. CONCLUSION: Total RNA can be successfully obtained from MFGs. The results suggest that these genes play a role in glucose absorption and lactose synthesis in bovine mammary epithelial cells during lactation. Also, these results provide light on the differential expression of these genes among distinct Holstein-Friesian cow breeds and Egyptian buffalo subspecies throughout various lactation phases.

Regulatory roles of dopamine D2 receptor in milk protein production and apoptosis in mammary epithelial cells.

Dopamine D2 receptors (D2Rs) play crucial roles in regulating diverse physiological functions of the central nervous system and peripheral organs. D2Rs are also expressed in mammary glands. However, which cell types express D2Rs and whether they are involved in milk production remains unclear. The present findings revealed that D2Rs are expressed in the apical regions of the lateral membranes of mammary epithelial cells (MECs) in lactating mice. We also investigated the effects of the D2R agonist bromocriptine and/or antagonist domperidone on intracellular cAMP levels, milk protein production, and apoptosis in a lactation culture model of MECs that produce major milk components like lactating MECs in vivo. We found that bromocriptine decreased intracellular cAMP levels, whereas domperidone dose-dependently neutralized this effect. Bromocriptine also inhibited casein and lactoferrin production and suppressed activities of STAT5 and glucocorticoid receptors (GRs). Domperidone neutralized the inhibition of casein production as well as STAT5 and GR inactivation induced by bromocriptine. Furthermore, D2R activation by bromocriptine induced apoptosis and inactivated ERK, a signaling molecule responsible for promoting cell proliferation and survival. Domperidone attenuated ERK inactivation and apoptosis induced by bromocriptine. These findings suggest that D2Rs play regulatory roles in milk protein production and apoptosis in MECs.

IKZF3/Aiolos H195Y mutation identified in a mouse model of B cell leukemia results in altered DNA binding and altered STAT5-dependent gene expression.

Precursor B cell acute lymphoblastic leukemia (Pre-B-ALL) arises from developing B cells and frequently involves mutations in genes encoding transcription factors. In this study, we investigated the function of mutations in the transcription factor IKZF3 (Aiolos), R137* and H195Y, discovered in a mouse model of pre-B-ALL. R137* IKZF3 mutation resulted in a truncated protein, while electrophoretic mobility shift assay showed that H195Y IKZF3 mutation resulted in a protein with altered DNA binding. 38B9 pre-B cell lines were generated expressing WT and H195Y IKZF3 proteins. Anti-IKZF3 ChIP-seq showed that H195Y IKZF3 interacted with a larger number of sites that were different than WT IKZF3. Treatment with interleukin-7 induced changes in gene expression in 38B9 cells expressing WT IKZF3, but did not induce any changes in gene expression in cells expressing H195Y IKZF3. Anti-STAT5 ChIP-seq showed that expression of H195Y IKZF3 resulted in redistribution of STAT5 binding sites in the genome. H195Y IKZF3 binding sites overlapped with a subset of STAT5 binding sites, including in the promoter of the Cish gene. These findings suggest that H195Y mutation of IKZF3 results in altered DNA binding specificity and altered binding of STAT5 to target genes.

Evidence of additive genetic variation for major milk proteins in dairy cows: A meta-analysis.

In the past, there have been reports of genetic parameters for milk proteins in various dairy cattle populations. The high variability among genetic parameter estimates has been caused by this. This study aimed to use a random-effects meta-analysis model to compile published estimates of genetic parameter for major milk proteins of α-lactalbumin, ß-lactoglobulin, sum of whey proteins, casein, αs1-casein, αs2-casein, ß-casein, and κ-casein in dairy cows. The study used a total of 140 heritability and 256 genetic correlation estimates from 23 papers published between 2004 and 2022. The estimated range of milk protein heritability is from 0.284 (for α-lactalbumin in milk) to 0.596 (for sum of whey proteins). The genetic correlation estimates between casein and milk yield, milk fat and protein percentages were -0.461, 0.693, and 0.976, respectively (p < 0.05). The genetic correlation estimates between milk proteins expressed as a percentage of milk were significant and varied from 0.177 (between ß-lactoglobulin and κ-casein) to 0.892 (between αs1-casein and αs2-casein). Moderate-to-high heritability estimates for milk proteins and their low genetic associations with milk yield and composition indicated the possibility for improving milk proteins in a genetic selection plan with negligible correlated effects on production traits in dairy cows.

A novel opsonic eCIRP inhibitor for lethal sepsis.

Sepsis is a life-threatening inflammatory condition partly orchestrated by the release of various damage-associated molecular patterns such as extracellular cold-inducible RNA-binding protein (eCIRP). Despite advances in understanding the pathogenic role of eCIRP in inflammatory diseases, novel therapeutic strategies to prevent its excessive inflammatory response are lacking. Milk fat globule-epidermal growth factor-VIII (MFG-E8) is critical for the opsonic clearance of apoptotic cells, but its potential involvement in the removal of eCIRP was previously unknown. Here, we report that MFG-E8 can strongly bind eCIRP to facilitate αvß3-integrin-dependent internalization and lysosome-dependent degradation of MFG-E8/eCIRP complexes, thereby attenuating excessive inflammation. Genetic disruption of MFG-E8 expression exaggerated sepsis-induced systemic accumulation of eCIRP and other cytokines, and consequently exacerbated sepsis-associated acute lung injury. In contrast, MFG-E8-derived oligopeptide recapitulated its eCIRP binding properties, and significantly attenuated eCIRP-induced inflammation to confer protection against sepsis. Our findings suggest a novel therapeutic approach to attenuate eCIRP-induced inflammation to improve outcomes of lethal sepsis.

Site specific insertion of a transgene into the murine α-casein (CSN1S1) gene results in the predictable expression of a recombinant protein in milk.

Gene loci of highly expressed genes provide ideal sites for transgene expression. Casein genes are highly expressed in mammals leading to the synthesis of substantial amounts of casein proteins in milk. The α-casein (CSN1S1) gene has assessed as a site of transgene expression in transgenic mice and a mammary gland cell line. A transgene encoding an antibody light chain gene (A1L) was inserted into the α-casein gene using sequential homologous and site-specific recombination. Expression of the inserted transgene is directed by the α-casein promoter, is responsive to lactogenic hormone activation, leads to the synthesis of a chimeric α-casein/A1L transgene mRNA, and secretion of the recombinant A1L protein into milk. Transgene expression is highly consistent in all transgenic lines, but lower than that of the α-casein gene (4%). Recombinant A1L protein accounted for 0.5% and 1.6% of total milk protein in heterozygous and homozygous transgenic mice, respectively. The absence of the α-casein protein in homozygous A1L transgenic mice leads to a reduction of total milk protein and delayed growth of the pups nursed by these mice. Overall, the data demonstrate that the insertion of a transgene into a highly expressed endogenous gene is insufficient to guarantee its abundant expression.

Single and longitudinal genome-wide association studies for dairy traits available in goats with three recorded lactations.

Milk yield and composition phenotypes are systematically recorded across several lactations in goats, but the majority of genome-wide association studies (GWAS) performed so far have rather ignored the longitudinal nature of such data. Here, we have used two different GWAS approaches to analyse data from three lactations recorded in Murciano-Granadina goats. In Analysis 1, independent GWAS have been carried out for each trait and lactation, while a single longitudinal GWAS, jointly considering all data, has been performed in Analysis 2. In both analyses, genome-wide significant QTL for lactose percentage on chromosome 2 (129.77-131.01 Mb) and for milk protein percentage on the chromosome 6 (74.8-94.6 Mb) casein gene cluster region were detected. In Analysis 1, several QTL were not replicated in all three lactations, possibly due to the existence of lactation-specific genetic determinants. In Analysis 2, we identified several genome-wide significant QTL related to milk yield and protein content that were not uncovered in Analysis 1. The increased number of QTL identified in Analysis 2 suggests that the longitudinal GWAS is particularly well suited for the genetic analysis of dairy traits. Moreover, our data confirm that variability within or close to the casein complex is the main genetic determinant of milk protein percentage in Murciano-Granadina goats.

Unravelling heterogeneous effects of cancer­associated fibroblasts on poor prognosis markers in breast cancer EM­G3 cell line: In vitro­targeted treatment (anti­IL-6, anti­VEGF-A, anti­MFGE8) based on transcriptomic profiling.

Breast cancer is the most frequently diagnosed cancer in women worldwide. Although dramatically increased survival rates of early diagnosed cases have been observed, late diagnosed patients and metastatic cancer may still be considered fatal. The present study's main focus was on cancer­associated fibroblasts (CAFs) which is an active component of the tumor microenvironment (TME) regulating the breast cancer ecosystem. Transcriptomic profiling and analysis of CAFs isolated from breast cancer skin metastasis, cutaneous basal cell carcinoma, and squamous cell carcinoma unravelled major gene candidates such as IL6, VEGFA and MFGE8 that induced co­expression of keratins­8/­14 in the EM­G3 cell line derived from infiltrating ductal breast carcinoma. Western blot analysis of selected keratins (keratin­8, ­14, ­18, ­19) and epithelial­mesenchymal transition­associated markers (SLUG, SNAIL, ZEB1, E­/N­cadherin, vimentin) revealed specific responses pointing to certain heterogeneity of the studied CAF populations. Experimental in vitro treatment using neutralizing antibodies against IL-6, VEGF­A and MFGE8 attenuated the modulatory effect of CAFs on EM­G3 cells. The present study provided novel data in characterizing and understanding the interactions between CAFs and EM­G3 cells in vitro. CAFs of different origins support the pro­inflammatory microenvironment and influence the biology of breast cancer cells. This observation potentially holds significant interest for the development of novel, clinically relevant approaches targeting the TME in breast cancer. Furthermore, its implications extend beyond breast cancer and have the potential to impact a wide range of other cancer types.

Cell factory-based milk protein biomanufacturing: Advances and perspectives.

The increasing global population and protein demand cause global challenges for food supply. Fueled by significant developments in synthetic biology, microbial cell factories are constructed for the bioproduction of milk proteins, providing a promising approach for scalable and cost-effective production of alternative proteins. This review focused on the synthetic biology-based microbial cell factory construction for milk protein bioproduction. The composition, content, and functions of major milk proteins were first summarized, especially for caseins, α-lactalbumin, and ß-lactoglobulin. An economic analysis was performed to determine whether cell factory-based milk protein production is economically viable for industrial production. Cell factory-based milk protein production is proved to be economically viable for industrial production. However, there still exist some challenges for cell factory-based milk protein biomanufacturing and application, including the inefficient production of milk proteins, insufficient investigation of protein functional property, and insufficient food safety evaluation. Constructing new high-efficiency genetic regulatory elements and genome editing tools, coexpression/overexpression of chaperone genes, and engineering protein secretion pathways and establishing a cost-effective protein purification method are possible ways to improve the production efficiency. Milk protein biomanufacturing is one of the promising approaches to acquiring alternative proteins in the future, which is of great importance for supporting cellular agriculture.

Analysis of the relationship between short tandem repeats and lactation performance of Xinjiang Holstein cows.

Microsatellite markers, also known as short tandem repeats (STRs), are important for marker-assisted selection to detect genetic polymorphism, and they are uniformly distributed in eukaryotic genomes. To analyze the relationship between microsatellite loci and lactation traits of Holstein cows in Xinjiang, 175 lactating cows with similar birth dates, the same parity, and similar calving dates were selected, and 10 STR loci closely linked to quantitative trait loci were used to analyze the correlation between each STR locus and four lactation traits (daily milk yield, milk fat percentage, milk protein percentage, and lactose percentage). All loci showed different degrees of genetic polymorphism. The average values of observed alleles, effective alleles, expected heterozygosity, observed heterozygosity, and polymorphic information content of the 10 STR loci were 10, 3.11, 0.62, 0.64, and 0.58, respectively. Chi-square and G-square tests showed that all populations of loci were in accordance with the Hardy-Weinberg equilibrium. Analysis of the correlation between STR locus genotype and lactation performance in the whole lactation period showed three loci (namely, BM143, BM415, and BP7) with no significant correlation with all lactation traits, two loci (BM302 and UWCA9) related to milk yield, three loci (BM103, BM302, and BM6425) related to milk fat percentage, two loci (BM302 and BM6425) related to milk protein percentage, and three loci (BM1443, BM302, and BMS1943) related to lactose percentage. The microsatellite loci selected in this study showed rich polymorphism in the experimental dairy cow population and were related to the lactation traits, which can be used for the evaluation of genetic resources and early breeding and improvement of Holstein dairy cows in Xinjiang.

mTORC2-AKT-LAT1 signalling participates in methionine-induced ß-CASEIN expression in mammary epithelial cells of dairy cows.

This study investigated the role of the mammalian target of rapamycin complex 2 (mTORC2)-protein kinase B (AKT) signalling in methionine (Met)-induced L-type amino acid transporter 1 (LAT1) expression and milk protein production. Primary mammary epithelial cells (MECs) from mammary parenchymal tissues of three lactating cows and MAC-T bovine MECs were cultured with or without 0.6 mM Met. Rapamycin-insensitive companion of mTOR (RICTOR) siRNA, the mTORC1 inhibitor rapamycin and the AKT activator SC79 were used to evaluate the effects of mTORC2-AKT signalling on Met-induced LAT1 expression and function. Each experiment was performed three times. Data were analysed with a two-sided unpaired t test or ANOVA with the Bonferroni multiple-comparison test. Western blotting showed that Met stimulation increased RICTOR expression (~244.67%; p < 0.05; control, 0.15 ± 0.026; Met, 0.517 ± 0.109) and AKT-S473 levels (~281.42%; p < 0.01; control, 0.253 ± 0.067; Met, 0.965 ± 0.019) in both primary MECs and MAC-T cells. Rapamycin-induced mTORC1 signalling inhibition decreased only Met-induced ß-CASEIN expression by ~21.24% (p < 0.01; Met, 0.777 ± 0.01; Met and rapamycin, 0.612 ± 0.04) and did not affect Met-stimulated AKT-S473 levels, suggesting that mTORC2-AKT activation upon Met stimulation also contributes to milk protein synthesis. LAT1 participates in Met-induced ß-CASEIN expression. In dairy cow MECs, mTORC2 inhibition by RICTOR siRNA decreased LAT1 levels on the plasma membrane by ~45.13% (p < 0.01; control, 0.359 ± 0.006; siRICTOR, 0.197 ± 0.004). However, SC79-induced AKT activation had the opposite effect (p < 0.01). In primary MECs and MAC-T cells, Met stimulation increased cytosolic and plasma membrane LAT1 expression respectively (MECs, 113.98% and 58.43%; MAC-T, 165.85% and 396.39%; p < 0.05). However, RICTOR siRNA significantly reduced Met-induced plasma membrane LAT1 expression (~76.48%; Met, 0.539 ± 0.05; Met and siRICTOR, 0.127 ± 0.012; p < 0.05). Thus, Met increased LAT1 expression and function via mTORC2-AKT signalling, upregulating milk protein synthesis in dairy cow MECs.

Detection of A2A2 genotype of beta casein protein (CSN2) gene in local, exotic and cross bred cattle in Pakistan.

Genetic variants of bovine Beta-casein protein (CSN2) gene especially A1 and A2 are the most important variants in dairy cattle. A1 milk protein is considered as risk factor for different disease and milk intolerance which release Beta-Casomorphin-7 during digestion which is a bioactive opioid but not released from A2 milk protein. This opioid is responsible for several human health problems like Coronary Heart disease, type 1 diabetics, milk intolerance and other neurological disorders. In present study, 360 blood sample were collected from Lohani, Achai, jersey, Holstein Friesian, Achai x jersey, Friesian x Sahiwal and Sahiwal x Friesian from different region of Khyber Pakhtunkhwa (KP) province. The polymerase chain reaction (PCR) amplicons were sequenced for the identification of polymorphism in exon 7 of Beta-casein protein (CSN2) gene. Sequencing analysis explored CSN2 genotype in exon 7 using the Genomic sequence from GenBank (X.71104) g.8101 C > A at codon 67. The allelic and genotypic frequencies of CSN2 gene were analyzed and observed that Holstein Friesian cattle exhibited A1A2 33%, A1A1 50% and A2A2 17%, Jersey cattle show 68% A1A1, 18% A1A2 and 14% A2A2, Sahiwal x Friesian 56% A1A1, 26% A1A2 and 18% A2A2, Jersey × Achai 78% A2A2, 15% A1A2 and 7% A1A1, Achai 100% A2A2 Lohani 100% A2A2. This is a preliminary study, conducted with meager resources, therefore, it is very difficult to make conclusion that which particular breed possess harmful alleles and which breed possess useful alleles of beta-casein gene. Therefore, a comprehensive molecular work is needed to be performed with greater number of samples sequencing.

Genetic variants of CSN1S1, CSN2, CSN3, and BLG genes and their association with dairy production traits in Sahiwal cattle and Nili-Ravi buffaloes.

Milk protein genes are associated with milk yield and composition in dairy animals. The present study aimed to identify milk protein genes (CSN1S1, CSN2, CSN3, and BLG) genetic variants and their association with milk yield in Sahiwal cattle and Nili-Ravi buffaloes. One hundred animals from each species were selected to collect blood samples and milk production records. Primers were designed for these milk protein genes for PCR amplification. Sequencing of resultant PCR products revealed a higher number of SNPs (13 vs. 7, 5 vs. 1, and 6 vs. 2) in Sahiwal as compared to Nili-Ravi animals in CSN1S1, CSN2, and CSN3 genes, respectively. However, a single SNP was observed in BLG gene of both species. Association analysis revealed that one SNP in BLG gene of Nili-Ravi was associated (p < 0.05) with 305-day milk yield. Two SNPs at CSN1S1 gene in Sahiwal were associated with dry-period. Similarly, one SNP at CSN1S1 and two SNPs at CSN3 gene showed significant association (p < 0.05) with average calving-interval in Sahiwal while two SNPs in CSN1S1 gene were associated (p < 0.05) with this trait in Nili-Ravi. These SNPs could be helpful as candidate variants for marker-assisted selection in cattle and buffaloes for improvement of lactation performance.

CSN1S1 and CSN3 gene variants in female Murrah buffaloes in the Brazilian Amazon.

The characteristics of milk are controlled by several genes, with emphasis on the four genes from casein, CSN1S1; CSN1S2; CSN2 and CSN3, which are responsible encoding of fractions the milk protein. The study of genetic variants in these genes, seek to investigate alleles, insertions or deletions, that can directly reflect on productive characteristics, indicating differences in milk quality, composition and yield. The CSN1S1 and CSN3 genes were analyzed in lactating Murrah buffaloes using nucleotide sequencing. An SNP was found in the amplified fragment of the CSN1S1 gene, located in nucleotide number 2,123 of the promoter region in position nt-258 (A/G). As for the CSN3 gene, two SNPs of exon number 4 were identified in codons 33 (ACC/ATC) and 34 (ACC/ACT) of the analyzed fragment. This study contributes to important associations between genetic variants and the desired characteristics of milk and its derivatives in future studies, because the variants found may be associated with the quality of milk, enabling genetic selection to be assisted by molecular markers, indicating a major advance that makes it possible to select animals early.

Inflammatory Role of Milk Fat Globule-Epidermal Growth Factor VIII in Age-Associated Arterial Remodeling.

Background Age-associated aortic remodeling includes a marked increase in intimal medial thickness (IMT), associated with signs of inflammation. Although aortic wall milk fat globule-epidermal growth factor VIII (MFG-E8) increases with age, and is associated with aortic inflammation, it is not known whether MFG-E8 is required for the age-associated increase in aortic IMT. Here, we tested whether MFG-E8 is required for the age-associated increase in aortic IMT. Methods and Results To determine the role of MFG-E8 in the age-associated increase of IMT, we compared aortic remodeling in adult (20-week) and aged (96-week) MFG-E8 (-/-) knockout and age matched wild-type (WT) littermate mice. The average aortic IMT increased with age in the WT from 50±10 to 70±20 µm (P<0.0001) but did not significantly increase with age in MFG-E8 knockout mice. Because angiotensin II signaling is implicated as a driver of age-associated increase in IMT, we infused 30-week-old MFG-E8 knockout and age-matched littermate WT mice with angiotensin II or saline via osmotic mini-pumps to determine whether MFG-E8 is required for angiotensin II-induced aortic remodeling. (1) In WT mice, angiotensin II infusion substantially increased IMT, elastic lamina degradation, collagen deposition, and the proliferation of vascular smooth muscle cells; in contrast, these effects were significantly reduced in MFG-E8 KO mice; (2) On a molecular level, angiotensin II treatment significantly increased the activation and expression of matrix metalloproteinase type 2, transforming growth factor beta 1, and its downstream signaling molecule phosphorylated mother against decapentaplegic homolog 2, and collagen type I production in WT mice; however, in the MFG-E8 knockout mice, these molecular effects were significantly reduced; and (3) in WT mice, angiotensin II increased levels of aortic inflammatory markers phosphorylated nuclear factor-kappa beta p65, monocyte chemoattractant protein 1, tumor necrosis factor alpha, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 molecular expression, while in contrast, these inflammatory markers did not change in knockout mice. Conclusions Thus, MFG-E8 is required for both age-associated proinflammatory aortic remodeling and also for the angiotensin II-dependent induction in younger mice of an aortic inflammatory phenotype observed in advanced age. Targeting MFG-E8 would be a novel molecular approach to curb adverse arterial remodeling.

Inframe insertion and splice site variants in MFGE8 associate with protection against coronary atherosclerosis.

Cardiovascular diseases are the leading cause of premature death and disability worldwide, with both genetic and environmental determinants. While genome-wide association studies have identified multiple genetic loci associated with cardiovascular diseases, exact genes driving these associations remain mostly uncovered. Due to Finland's population history, many deleterious and high-impact variants are enriched in the Finnish population giving a possibility to find genetic associations for protein-truncating variants that likely tie the association to a gene and that would not be detected elsewhere. In a large Finnish biobank study FinnGen, we identified an association between an inframe insertion rs534125149 in MFGE8 (encoding lactadherin) and protection against coronary atherosclerosis. This variant is highly enriched in Finland, and the protective association was replicated in meta-analysis of BioBank Japan and Estonian biobank. Additionally, we identified a protective association between splice acceptor variant rs201988637 in MFGE8 and coronary atherosclerosis, independent of the rs534125149, with no significant risk-increasing associations. This variant was also associated with lower pulse pressure, pointing towards a function of MFGE8 in arterial aging also in humans in addition to previous evidence in mice. In conclusion, our results suggest that inhibiting the production of lactadherin could lower the risk for coronary heart disease substantially.

Chondroprotective Effects of Periodontal Ligament-Derived Stem Cells Conditioned Medium on Articular Cartilage After Impact Injury.

Paracrine factors secreted in the conditioned media (CMs) of periodontal ligament-derived stem cells (PDLSCs) have been shown to downregulate inflammatory effects of interleukin (IL)-1ß on chondrocytes wherein milk fat globule-epidermal growth factor 8 (MFG-E8) is one of the PDLSCs' highly secretory proteins. Therefore, the objective of this study was to investigate the ability of PDLSC CMs and MFG-E8 to reduce the inflammatory effects of impact injury on porcine talar articular cartilage (AC) and IL-1ß on chondrocytes, respectively. Stem cells were isolated from human periodontal ligaments. The MFG-E8 content in CM collected at 5% and 20% oxygen was measured by ELISA assay and compared across subcultures and donors. AC samples were divided into three groups: control, impact, and impact+CM. Chondrocytes were isolated from pig knees and were divided into three groups: control, IL-1ß, and IL-1ß+MFG-E8. Gene expression data were analyzed by reverse transcription-polymerase chain reaction. It was found that impact load and IL-1ß treatment upregulated IL-1ß, TNF-α, ADAMTS-4, and ADAMTS-5 gene expression in AC and chondrocytes, respectively. PDLSCs-CM prevented the upregulation of all four genes due to impact, whereas MFG-E8 prevented upregulation of IL-1ß, ADAMTS-4, and ADAMTS-5 in chondrocytes, but it did not prevent TNF-α upregulation. There were no significant differences in MFG-E8 content in CM among oxygen levels, passage numbers, or donors. The findings suggested that MFG-E8 is an effective anti-inflammatory agent contributing to the chondroprotective effects of PDLSCs-CM on acutely injured AC. Thus, introducing PDLSCs-CM to sites of acute traumatic AC injury could prevent the development of post-traumatic osteoarthritis.

Evaluation of the α-casein (CSN1S1) locus as a potential target for a site-specific transgene integration.

Transgenic animals are an important tool in biotechnology, including the production of recombinant proteins in the milk. Traditionally, expression constructs are based on hybrid vectors bearing mammary gland specific regulatory elements from the α-casein (Csn1s1), ß-casein (Csn2), whey acidic protein (WAP), or ß-lactoglobulin (BLG) genes. Overexpression from the randomly integrated vectors typically provides high levels of expression, but has drawbacks due to unpredictable genome localization. CRISPR-Cas9 targeted transgene integration into the endogenous casein locus could alleviate the need for extensive animal screening to achieve high and reproducible expression levels. We decided to evaluate such a "precise" integration approach, placing the human granulocyte-macrophage colony-stimulating factor (hGMCSF) gene under control of the mouse endogenous alpha-S1-casein (Csn1s1) promoter. We designed two types of transgene integrations: a knock-in in the second exon of the Csn1s1 (INS-GM) and a full-size Csn1s1 replacement with hGMCSF (REP-GM) which was never tested before. The INS-GM approach demonstrated low transgene expression and milk protein levels (0.4% of Csn2 transcripts; 2-11 µg/ml hGMCSF). This was probably caused by the absence of the 3'-polyadenylation signal in the hGMCSF transgene. REP-GM animals displayed high transgene expression, reaching and slightly exceeding the level of the endogenous Csn1s1 (30-40% of Csn2 transcripts), but yielded less hGMCSF protein than expected (0.2-0.5 mg/ml vs 25 mg/ml of Csn1s1), indicating that translation of the protein is not optimal. Homozygous inserts leading to the Csn1s1 knock-out did not have any long standing effects on the animals' health. Thus, in our experimental design, site-specific transgene integration into the casein locus did not provide any significant advantage over the overexpression approach.