Adipocyte hypertrophy and lipid dynamics underlie mammary gland remodeling after lactation.

Adipocytes undergo pronounced changes in size and behavior to support diverse tissue functions, but the mechanisms that control these changes are not well understood. Mammary gland-associated white adipose tissue (mgWAT) regresses in support of milk fat production during lactation and expands during the subsequent involution of milk-producing epithelial cells, providing one of the most marked physiological examples of adipose growth. We examined cellular mechanisms and functional implications of adipocyte and lipid dynamics in the mouse mammary gland (MG). Using in vivo analysis of adipocyte precursors and genetic tracing of mature adipocytes, we find mature adipocyte hypertrophy to be a primary mechanism of mgWAT expansion during involution. Lipid tracking and lipidomics demonstrate that adipocytes fill with epithelial-derived milk lipid. Furthermore, ablation of mgWAT during involution reveals an essential role for adipocytes in milk trafficking from, and proper restructuring of, the mammary epithelium. This work advances our understanding of MG remodeling and tissue-specific roles for adipocytes.

Increased epigenetic age in normal breast tissue from luminal breast cancer patients.

BACKGROUND: Age is one of the most important risk factors for developing breast cancer. However, age-related changes in normal breast tissue that potentially lead to breast cancer are incompletely understood. Quantifying tissue-level DNA methylation can contribute to understanding these processes. We hypothesized that occurrence of breast cancer should be associated with an acceleration of epigenetic aging in normal breast tissue. RESULTS: Ninety-six normal breast tissue samples were obtained from 88 subjects (breast cancer = 35 subjects/40 samples, unaffected = 53 subjects/53 samples). Normal tissue samples from breast cancer patients were obtained from distant non-tumor sites of primary mastectomy specimens, while samples from unaffected women were obtained from the Komen Tissue Bank (n = 25) and from non-cancer-related breast surgery specimens (n = 28). Patients were further stratified into four cohorts: age < 50 years with and without breast cancer and age ≥ 50 with and without breast cancer. The Illumina HumanMethylation450k BeadChip microarray was used to generate methylation profiles from extracted DNA samples. Data was analyzed using the "Epigenetic Clock," a published biomarker of aging based on a defined set of 353 CpGs in the human genome. The resulting age estimate, DNA methylation age, was related to chronological age and to breast cancer status. The DNAmAge of normal breast tissue was strongly correlated with chronological age (r = 0.712, p < 0.001). Compared to unaffected peers, breast cancer patients exhibited significant age acceleration in their normal breast tissue (p = 0.002). Multivariate analysis revealed that epigenetic age acceleration in the normal breast tissue of subjects with cancer remained significant after adjusting for clinical and demographic variables. Additionally, smoking was found to be positively correlated with epigenetic aging in normal breast tissue (p = 0.012). CONCLUSIONS: Women with luminal breast cancer exhibit significant epigenetic age acceleration in normal adjacent breast tissue, which is consistent with an analogous finding in malignant breast tissue. Smoking is also associated with epigenetic age acceleration in normal breast tissue. Further studies are needed to determine whether epigenetic age acceleration in normal breast tissue is predictive of incident breast cancer and whether this mediates the risk of chronological age on breast cancer risk.

A 3D Mammometric Comparison of Implant-Based Breast Reconstruction With and Without Acellular Dermal Matrix (ADM).

This retrospective study utilizes 3D imaging and mammometrics to compare implant-based breast reconstruction with and without the use of ADM. Previous studies have suggested improved aesthetic outcomes with the use of ADM, but none have been able to quantify this difference. Images were obtained at early and late time points following the expander-implant exchange procedure. Measurements included the point of maximum projection, the superior, inferior, medial and lateral volumetric distribution, and the distance from the point of maximum projection to the inframammary fold along the breast meridian. The patients' demographic information, implant size, and complication rate between the two cohorts were similar. In the early post-operative period, the patients with ADM demonstrated higher medial pole volume; however, this difference did not persist in the late post-operative period. Patients with ADM demonstrated a small but statistically significant greater point of maximum projection and length of lower pole curvature in comparison with the non-ADM cohort. In summary, the results of this study demonstrate improved mammometric measurements when ADM is used in implant-based breast reconstruction, supporting superior aesthetic outcomes in early and late post-operative time points. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Endothelial APLNR regulates tissue fatty acid uptake and is essential for apelin's glucose-lowering effects.

Treatment of type 2 diabetes mellitus continues to pose an important clinical challenge, with most existing therapies lacking demonstrable ability to improve cardiovascular outcomes. The atheroprotective peptide apelin (APLN) enhances glucose utilization and improves insulin sensitivity. However, the mechanism of these effects remains poorly defined. We demonstrate that the expression of APLNR (APJ/AGTRL1), the only known receptor for apelin, is predominantly restricted to the endothelial cells (ECs) of multiple adult metabolic organs, including skeletal muscle and adipose tissue. Conditional endothelial-specific deletion of Aplnr (AplnrECKO ) resulted in markedly impaired glucose utilization and abrogation of apelin-induced glucose lowering. Furthermore, we identified inactivation of Forkhead box protein O1 (FOXO1) and inhibition of endothelial expression of fatty acid (FA) binding protein 4 (FABP4) as key downstream signaling targets of apelin/APLNR signaling. Both the Apln-/- and AplnrECKO mice demonstrated increased endothelial FABP4 expression and excess tissue FA accumulation, whereas concurrent endothelial Foxo1 deletion or pharmacologic FABP4 inhibition rescued the excess FA accumulation phenotype of the Apln-/- mice. The impaired glucose utilization in the AplnrECKO mice was associated with excess FA accumulation in the skeletal muscle. Treatment of these mice with an FABP4 inhibitor abrogated these metabolic phenotypes. These findings provide mechanistic insights that could greatly expand the therapeutic repertoire for type 2 diabetes and related metabolic disorders.

A Unique Case of Allogeneic Fat Grafting Between Brothers.

We present a case of a 65-year-old man with cutaneous T-cell lymphoma treated with radiation therapy and an allogeneic hematopoietic stem cell transplant from his human leukocyte antigen-matched brother. Engraftment was successful, but the patient went on to develop painful, radiation-induced ulcers. The ulcers were fat-allografted using liposuctioned fat from his brother because of the patient's unique chimeric state. Postprocedure follow-up revealed epithelialization of the ulcer sites and significant improvement in neuropathic pain. Our unique case study supports the use of fat grafting for its restorative purposes and for its ability to alleviate chronic neuropathic pain. Additionally, it appears that our case provides a basis of a general approach to the treatment of radiation-induced ulcers in chimeric patients with lymphoid malignancies.

In Vivo Changes of Breast Perfusion After Augmentation.

BACKGROUND: Revision surgeries after breast augmentation are associated with an increased risk of complications (eg, nipple areolar complex [NAC]) necrosis. Consequently, maintaining perfusion to the NAC is a critical aspect of secondary breast surgery. OBJECTIVES: The purpose of this study was to examine in vivo changes in perfusion to the NAC after implant breast augmentation using magnetic resonance imaging (MRI) technology. METHODS: High-resolution 3 Tesla MRI images of 10 women (20 breasts) with previous breast augmentation were compared to a control population of 15 women (30 breasts). Perforators from the internal mammary artery and lateral thoracic artery were examined for the diameter of the originating perforator, distance between the nipple and most distally visualized point of the medial and lateral perforator, and dominance pattern between the medial vs lateral perforators. RESULTS: No difference was found in the caliber of the medial vessels in the implant group compared to the control group. In contrast, the caliber of the lateral blood vessels trended towards being 20% larger in diameter in the augmented breasts. The distances between the nipple and the medial and lateral vessels increased. The frequencies in the distribution of dominance were not significantly different between the implant group and the control group. CONCLUSIONS: Overall, medial and lateral blood supply to the NAC are preserved in the augmented patient. Our results suggest a slight delay effect that seems to increase the caliber of the lateral perforators. In addition, the tissue expansion provided by the implants effectively increases the length of both perforators. LEVEL OF EVIDENCE: 3 Therapeutic.

SREBP-1c/MicroRNA 33b Genomic Loci Control Adipocyte Differentiation.

White adipose tissue (WAT) is essential for maintaining metabolic function, especially during obesity. The intronic microRNAs miR-33a and miR-33b, located within the genes encoding sterol regulatory element-binding protein 2 (SREBP-2) and SREBP-1, respectively, are transcribed in concert with their host genes and function alongside them to regulate cholesterol, fatty acid, and glucose metabolism. SREBP-1 is highly expressed in mature WAT and plays a critical role in promoting in vitro adipocyte differentiation. It is unknown whether miR-33b is induced during or involved in adipogenesis. This is in part due to loss of miR-33b in rodents, precluding in vivo assessment of the impact of miR-33b using standard mouse models. This work demonstrates that miR-33b is highly induced upon differentiation of human preadipocytes, along with SREBP-1. We further report that miR-33b is an important regulator of adipogenesis, as inhibition of miR-33b enhanced lipid droplet accumulation. Conversely, overexpression of miR-33b impaired preadipocyte proliferation and reduced lipid droplet formation and the induction of peroxisome proliferator-activated receptor γ (PPARγ) target genes during differentiation. These effects may be mediated by targeting of HMGA2, cyclin-dependent kinase 6 (CDK6), and other predicted miR-33b targets. Together, these findings demonstrate a novel role of miR-33b in the regulation of adipocyte differentiation, with important implications for the development of obesity and metabolic disease.

Cadaveric study of breast measurements during augmentation with implants.

BACKGROUND: The goal of this study was to compare immediate changes in breast shape and nipple position using different implant shapes and volumes, with variable release of the pectoralis major muscle in fresh cadavers. METHODS: Seventeen fresh cadaveric breasts were analyzed. Six different augmentation procedures were performed, including pocket dissection in the submuscular and subglandular planes, and partial and full release of the pectoralis major muscle insertion. Round and contoured implant volumes of 200, 300, 400, 500, and 600 ml were used, resulting in a total of 30 procedures per breast. RESULTS: Projection was greater when subjects received contoured implants versus round implants in all volumes both in submuscular and in subglandular pockets (p < 0.001). For implants larger than 200 ml, projection was greater when a subglandular pocket was chosen (p < 0.02), for both round and contoured implants. Nipple-to-inframammary fold distance was increased with contoured implants compared with round implants in both subglandular and submuscular pockets for all implant volumes (p < 0.05). CONCLUSIONS: Implant shape, volume, and pocket location influence projection, causing it to increase in a linear fashion. Partial pectoralis major release seems to affect projection only for small volumes, but does not influence nipple position in the immediate setting. As projection increases with augmentation volume, cephalad movement of the nipple is produced by a relative increase in nipple-to-inframammary fold distance compared with the midclavicular point-to-nipple distance in our cadaveric sample.

A tissue quality index: an intrinsic control for measurement of effects of preanalytical variables on FFPE tissue.

While efforts are made to improve tissue quality and control preanalytical variables, pathologists are often confronted with the challenge of molecular analysis of patient samples of unknown quality. Here we describe a first attempt to construct a tissue quality index (TQI) or an intrinsic control that would allow a global assessment of protein status based on quantitative measurement of a small number of selected, informative epitopes. Quantitative immunofluorescence (QIF) of a number of proteins was performed on a series of 93 breast cancer cases where levels of expression were assessed as a function of delayed time to formalin fixation. A TQI was constructed based on the combination of proteins that most accurately reflect increased and decreased levels of expression in proportion to delay time. The TQI, defined by combinations of measurements of cytokeratin, ERK1/2 and pHSP-27 and their relationship to cold ischemic time were validated on a second build of the training series and on two independent breast tissue cohorts with recorded time to formalin fixation. We show an association of negative TQI values (an indicator for loss of tissue quality) with increasing cold ischemic time on both validation cohorts and an association with loss of ER expression levels on all three breast cohorts. Using expression levels of three epitopes, we can begin to assess the likelihood of delayed time to fixation or decreased tissue quality. This TQI represents a proof of concept for the use of epitope expression to provide a mechanism for monitoring tissue quality.

Radiation therapy and expander-implant breast reconstruction: an analysis of timing and comparison of complications.

BACKGROUND: The optimal timing of expander-implant exchange in the setting of postmastectomy radiation therapy (PMRT) remains unclear with prior reports yielding inconsistent and variable results. The purpose of this study was to characterize complications associated with the sequencing of expander-implant breast reconstruction before or after PMRT and to compare the outcomes between early (<4 months) and late (>4 months) expander-implant exchange in the subset of patients who received PMRT before exchange. MATERIALS AND METHODS: The medical records of all patients PMRT in the setting of tissue expander-implant breast reconstruction between June 2004 and June 2011 at our institution were reviewed retrospectively. Patients were first classified as having undergone expander-implant exchange before the initiation of PMRT or after the completion of PMRT. Patients who underwent expander-implant exchange after PMRT were then classified as having undergone exchange early (<4 months after PMRT) or late (>4 months after PMRT). All complications requiring additional surgery or hospitalization were recorded. RESULTS: Fifty-five eligible patients were identified as having undergone 56 two-stage tissue expander-implant breast reconstructions. Twenty-two reconstructions underwent exchange before PMRT and 34 reconstructions underwent exchange after PMRT. There was no significant difference in overall complication rate (54.55% vs 47.06%, P = 0.785) or reconstruction failure rate (13.64% vs 20.59%, P = 0.724) between the 2 cohorts. Twenty reconstructions underwent exchange less than 4 months after PMRT and 14 underwent exchange more than 4 months after PMRT. There was no significant difference in overall complication rate (40% vs 57.14%, P = 0.487) or failure rate (25% vs 14.29%, P = 0.672) between the 2 groups. Trends suggest a higher rate of infection in patients who underwent exchange earlier (30% vs 14.29%, P = 0.422) and a higher rate of capsular contracture in patients who underwent exchange later (5% vs 21.43%, P = 0.283); however, statistical significance was not reached. CONCLUSIONS: Our findings suggest that neither the sequencing nor timing of expander-implant exchange in the setting of PMRT affects overall complication or reconstruction failure rate. However, the timing of exchange may impact the type of complication encountered. Further investigation is necessary to determine an optimal time for expander-implant exchange.

Silver-impregnated vacuum-assisted closure in the treatment of recalcitrant venous stasis ulcers.

Vacuum-assisted closure (VAC) has made a significant contribution to the treatment of acute and chronic wounds. Microdeformational forces from the VAC device accelerate granulation tissue formation when compared with moist saline dressing changes. We present 2 patients with multiple comorbid conditions and complex venous stasis ulcers that had persistent purulent drainage after conventional treatment modalities. Only after utilizing silver-impregnated VAC therapy (GranuFoam Silver), combining the antimicrobial benefits of silver with the advantages of VAC technology, were the wound beds adequately prepared for substantial split-thickness skin grafts. Based on these cases, the silver-impregnated VAC device may be a useful adjunct in wound bed preparation when standard therapies have failed to clear infected wounds. This may lead to improved healing rates and overall decreased wound burden in these complex patients.

Gene expression changes evoked in a venous segment exposed to arterial flow.

OBJECTIVE: This study was conducted to characterize the coordinated molecular changes evoked in the structure and composition of the wall of a venous segment when exposed to fistula flow. METHODS: An arteriovenous shunt was created in adult C57BL/6J mice. Remodeled veins and contralateral control jugular veins were isolated 7 days after surgery. Total RNA was isolated, linearly amplified, and the transcriptional profiles of this early adaptive response were obtained by microarray analysis. Histologic and immunohistochemical analyses were performed on remodeled veins and control veins isolated on days 1, 3, 5, and 7 after surgery to further examine distinct spatial and temporal aspects of this early process. RESULTS: There were 131 significantly upregulated and 165 downregulated genes in the remodeled vein compared with the control jugular vein. Genes involved in extracellular matrix reorganization were highly upregulated. Movat's pentachrome staining revealed ground substance on day 3 that was not observed on day 5. The appearance of elastin fibers was first observed on day 7. Morphometric analysis demonstrated maximum wall thickness on day 3. Immunohistochemical analysis revealed the presence of tenascin-C, thrombospondin, lysyl oxidase, and osteopontin in different cell types at different time points throughout the first week after surgery. CONCLUSION: Major changes in the organization of the extracellular matrix occur during the early response of venous remodeling. Elastin, tenascin-C, thrombospondin, lysyl oxidase, and osteopontin are expressed within the wall of the remodeling vein resulting in the de novo formation of an extracellular matrix scaffold that may be part of a critical adaptation program being evoked to allow the vessel to cope with its new biomechanical environment. CLINICAL RELEVANCE: The Kidney Dialysis Outcomes Quality Initiative has proposed the construction of arteriovenous fistulas as the primary vascular access for hemodialysis. As the vein is exposed to arterial flow, the vein wall dilates and a vascular remodeling process is triggered. With continued exposure, intimal hyperplasia occurs at the anastomosis that in many cases leads to failure. However, the molecular mechanisms by which the outflow vein remodels into a mature fistula remain incompletely understood. By investigating venous remodeling in a fistula model, candidate genes important for the remodeling process are discovered and their functional significance examined. Thus, the identification of relevant genes involved in this process should provide insight into arteriovenous fistula maturation and may suggest novel approaches for achieving higher patency rates.

Mastopexy with autologous augmentation after massive weight loss: the intercostal artery perforator (ICAP) flap.

The reconstruction of breast and upper-body deformities in massive weight loss (MWL) patients presents specific challenges to the plastic surgeon. In addition to significant breast ptosis and loss of breast volume, bariatric patients also have excessive lateral axillary and posterior truncal tissue that may require dermolipectomy for correction. A wise-pattern mastopexy was designed with a pedicled fasciocutaneous flap based on the intercostal artery perforators (ICAP) to correct breast ptosis, to restore breast volume, and to eliminate redundant upper truncal tissue. Five MWL patients underwent mastopexy with ICAP flap augmentation. All patients had stable and esthetically pleasing results 1, 3, and 6 months postoperatively. There were no complications of infection, wound dehiscence, seroma, or hematoma. Furthermore, there was no evidence of flap loss or tissue necrosis. Mastopexy with autologous augmentation using the ICAP flap was found to be a reliable method of breast reconstruction in the MWL patient.

Microdeformational wound therapy: effects on angiogenesis and matrix metalloproteinases in chronic wounds of 3 debilitated patients.

The vacuum-assisted closure (VAC) device causes microdeformations of the wound surface in contact with the foam. Because angiogenesis and matrix metalloproteinase (MMP) activity are altered in chronic wounds, we hypothesized that microdeformations stimulate capillary formation and affect MMP activity. A VAC device was used to deliver microdeformational wound therapy (MDWT) to the chronic wounds of 3 debilitated patients. Debrided tissue was obtained from wound areas with and without foam contact. Microvessel density and MMP activity were determined by immunohistochemistry and zymography, respectively. Microvessel density of MDWT-treated wounds was 4.5% (+/-0.8) compared with areas not covered by foam [1.6% (+/-0.1)] (P = 0.05) during the first week of treatment and 2.7% (+/-0.3) compared with untreated tissue [1.3% (+/-0.1)] (P = 0.03) during the second treatment week. Wounds subjected to MDWT had greater microvessel density compared with the same wound prior to treatment [1.5% (+/-0.3)] (P = 0.02). MMP-9/NGAL (neutrophil gelatinase-associated lipocalin), MMP-9, latent MMP-2, and active MMP-2 were reduced by 15%-76% in MDWT-treated wounds. MDWT provides a favorable wound-healing environment by increasing angiogenesis and decreasing MMP activity in chronic wounds.

Early adaptive responses of the vascular wall during venous arterialization in mice.

Venous arterialization occurs when a vein segment is transposed as a bypass graft into the arterial circulation, resulting in a structural and functional reorganization of the vascular wall in response to the new local biomechanical environment. Although the anatomical changes of venous arterialization have been well characterized, the molecular mechanisms of vascular remodeling remain incompletely understood. Here, we present a novel model of venous arterialization in mice wherein the external jugular vein is connected to the common carotid artery. The hemodynamic characteristics of the arterialized vein, as assessed by ultrasound and magnetic resonance imaging, resemble features of the arterial circulation. Temporal analyses of the morphological changes in the venous segment at 1, 3, and 7 days after surgery demonstrate preservation of the endothelium at all time points and formation of multiple smooth muscle layers by day 7. Expression of endothelial E-selectin and VCAM-1 was documented at early time points, concomitant with the presence of neutrophils and monocytes/macrophages in the vascular wall. In addition, endothelium-dependent permeability was decreased in the arterialized vein when compared to the contralateral control vein. Thus, this novel mouse model of venous arterialization displays anatomical and cellular features present in other species, and should help to characterize the molecular mechanisms of this adaptive response of the vascular wall to changes in its biomechanical environment.