Identification and Re-consent of Existing Cord Blood Donors for Creation of Induced Pluripotent Stem Cell Lines for Potential Clinical Applications.

We aim to create a bank of clinical grade cord blood-derived induced pluripotent stem cell lines in order to facilitate clinical research leading to the development of new cellular therapies. Here we present a clear pathway toward the creation of such a resource, within a strong quality framework, and with the appropriate regulatory, government and ethics approvals, along with a dynamic follow-up and re-consent process of cord blood donors from the public BMDI Cord Blood Bank. Interrogation of the cord blood bank inventory and next generation sequencing was used to identify and confirm 18 donors with suitable HLA homozygous haplotypes. Regulatory challenges that may affect global acceptance of the cell lines, along with the quality standards required to operate as part of a global network, are being met by working in collaboration with bodies such as the International Stem Cell Banking Initiative (ISCBI) and the Global Alliance for iPSC Therapies (GAiT). Ethics approval was granted by an Institutional Human Research Ethics Committee, and government approval has been obtained to use banked cord blood for this purpose. New issues of whole-genome sequencing and the relevant donor safeguards and protections were considered with input from clinical genetics services, including the rights and information flow to donors, and commercialization aspects. The success of these processes has confirmed feasibility and utility of using banked cord blood to produce clinical-grade iPSC lines for potential cellular therapies.

An adipogenic gel for surgical reconstruction of the subcutaneous fat layer in a rat model.

'Off-the-shelf' tissue-engineered skin alternatives for epidermal and dermal skin layers are available; however, no such alternative for the subdermal fat layer exists. Without this well-vascularized layer, skin graft take is variable and grafts may have reduced mobility, contracture and contour defects. In this study a novel adipose-derived acellular matrix (Adipogel) was investigated for its properties to generate subdermal fat in a rat model. In a dorsal thoracic site, a 1 × 1 cm Adipogel implant was inserted within a subdermal fat layer defect. In a dorsal lumbar site, an Adipogel implant was inserted in a subfascial pocket. Contralateral control defects remained empty. At 8 weeks wound/implant sites were evaluated histologically, immunohistochemically and morphometrically. Identifiable thoracic Adipogel implants lost volume in vivo over 8 weeks. Neovascularization and adipogenesis were evident within implants and adipocyte percentage volume was 33.07 ± 6.55% (mean ± SEM). A comparison of entire cross-sections of thoracic wounds demonstrated a significant increase in total wound fat in Adipogel-implanted wounds (37.19 ± 4.48%, mean ± SEM) compared to control (16.53 ± 4.60%; p = 0.0092), indicating that some Adipogel had been completely converted to normal fat. At the lumbar site, Adipogel also lost volume, appearing flattened, although fat generation and angiogenesis occurred. At both sites macrophage infiltration was mild, whilst many infiltrating cells were PDGFRß-positive mesenchymal cells. Adipogel is adipogenic and angiogenic and is a promising candidate for subcutaneous fat regeneration; it has the potential to be a valuable adjunct to wound-healing therapy and reconstructive surgery practice. Copyright © 2015 John Wiley & Sons, Ltd.

A Streamlined Method for the Preparation of Growth Factor-enriched Thermosensitive Hydrogels from Soft Tissue.

Hydrogels are an ideal medium for the expansion of cells in three dimensions. The ability to induce cell expansion and differentiation in a controlled manner is a key goal in tissue engineering. Here we describe a detailed method for producing hydrogels from soft tissues with an emphasis on adipose tissue. In this method, soluble, extractable proteins are recovered from the tissue and stored while the remaining insoluble tissue is processed and solubilised. Once the tissue has been sufficiently solubilised, the extracted proteins are added. The resulting product is a thermosensitive hydrogel with proteins representative of the native tissue. This method addresses common issues encountered when working with some biomaterials, such as high lipid content, DNA contamination, and finding an appropriate sterilisation method. Although the focus of this article is on adipose tissue, using this method we have produced hydrogels from other soft tissues including muscle, liver, and cardiac tissue.

Extracellular matrix fibronectin mediates an endothelial cell response to shear stress via the heparin-binding, matricryptic RWRPK sequence of FNIII1H.

Endothelial cells (EC) respond to mechanical forces such as shear stress in a variety of ways, one of which is cytoskeletal realignment in the direction of flow. Our earlier studies implicated the extracellular matrix protein fibronectin in mechanosensory signaling to ECs in intact arterioles, via a signaling pathway dependent on the heparin-binding region of the first type III repeat of fibrillar fibronectin (FNIII1H). Here we test the hypothesis that FNIII1H is required for EC stress fiber realignment under flow. Human umbilical vein ECs (HUVECs) exposed to defined flow conditions were used as a well-characterized model of this stress fiber alignment response. Our results directly implicate FNIII1H in realignment of stress fibers in HUVECs and, importantly, show that the matricryptic heparin-binding RWRPK sequence located in FNIII1 is required for the response. Furthermore, we show that flow-mediated stress fiber realignment in ECs adhered via α5ß1-integrin-specific ligands does not occur in the absence of FHIII1H, whereas, in contrast, αvß3-integrin-mediated stress fiber realignment under flow does not require FNIII1H. Our findings thus indicate that there are two separate mechanosignaling pathways mediating the alignment of stress fibers after exposure of ECs to flow, one dependent on αvß3-integrins and one dependent on FNIII1H. This study strongly supports the conclusion that the RWRPK region of FNIII1H may have broad capability as a mechanosensory signaling site.

Macrophage phenotype in response to implanted synthetic scaffolds: an immunohistochemical study in the rat.

Macrophages predominate among the cells that directly interact with biomaterials and are key orchestrators of host-biomaterial interactions. However, the macrophage response to synthetic scaffolds in particular has not been well studied. The aim of this study was therefore to characterise the macrophage response to several synthetic scaffolds in the rat using immunohistological techniques for a panel of markers of macrophage subclass or activation, including ED1 (CD68), ED2 (CD163), CD80, mannose receptor and inducible nitric oxide synthase (iNOS). Materials were implanted subcutaneously and collected after 6-8 weeks during the chronic phase of the host response. Unmodified polycaprolactone scaffolds uniquely demonstrated a total lack of both macrophage adherence to surfaces and a wider foreign body response compared to scaffolds composed of poly(lactic-co-glycolic acid) (PLGA) and polyurethanes (PURs), with those macrophages present having a clear M2 (MR+, CD80-, iNOS-) phenotype. PLGA scaffolds displayed an M1-dominant (CD80+, iNOS+, MR-) response with substantial foreign body giant cell (FBGC) formation, whilst PUR scaffold FBGCs had a more mixed M1 (CD80+, iNOS+) and M2 (MR+) phenotype. The study also identified that the use of the ED1 antibody in the rat as a pan-macrophage marker is problematic as there is a separate and substantial ED2-positive macrophage population that it does not label, both in response to biomaterials and in normal tissues. The biomaterial-dependent nature of activation for both macrophages and FBGCs was confirmed, and nuanced M1/M2 phenotypes were described.

Highly porous and mechanically robust polyester poly(ethylene glycol) sponges as implantable scaffolds.

The development of suitable scaffolds plays a significant role in tissue engineering research. Although scaffolds with promising features have been produced via a variety of innovative methods, there are no fully synthetic tissue engineering scaffolds that possess all the desired properties in one three-dimensional construct. Herein, we report the development of novel polyester poly(ethylene glycol) (PEG) sponges that display many of the desirable scaffold characteristics. Our novel synthetic approach utilizes acidchloride/alcohol chemistry, whereby the reaction between a hydroxyl end-functionalized 4-arm PEG and sebacoyl chloride resulted in cross-linking and simultaneous hydrogen chloride gas production, which was exploited for the in situ formation of highly interconnected pores. Variation of the fabrication conditions, including the precursor volume and concentration, allowed the pore size and structure as well as the compressive properties to be tailored. The sponges were found to possess excellent elastic properties, preserving their shape even after 80% compressive strain without failure. The benign properties of the sponges were demonstrated in an in vivo subcutaneous rat model, which also revealed uniform infiltration of vascularized tissue by 8 weeks and complete degradation of the sponges by 16 weeks, with only a minimal inflammatory response being observed over the course of the experiments.

Hypoxic conditioning enhances the angiogenic paracrine activity of human adipose-derived stem cells.

Human adipose-derived stem cells (ASCs) secrete cytokines and growth factors that can be harnessed in a paracrine fashion for promotion of angiogenesis, cell survival, and activation of endogenous stem cells. We recently showed that hypoxia is a powerful stimulus for an angiogenic activity from ASCs in vitro and here we investigate the biological significance of this paracrine activity in an in vivo angiogenesis model. A single in vitro exposure of ASCs to severe hypoxia (<0.1% O2) significantly increased both the transcriptional and translational level of the vascular endothelial growth factor-A (VEGF-A) and angiogenin (ANG). The angiogenicity of the ASC-conditioned medium (ASC(CM)) was assessed by implanting ASC(CM)-treated polyvinyl alcohol sponges subcutaneously for 2 weeks in mice. The morphometric analysis of anti-CD31-immunolabeled sponge sections demonstrated an increased angiogenesis with hypoxic ASC(CM) treatment compared to normoxic control ASC(CM) treatment (percentage vascular volume; 6.0%±0.5% in the hypoxic ASC(CM) vs. 4.1%±0.7% in the normoxic ASC(CM), P<0.05). Reduction of VEGF-A and ANG levels in the ASC(CM) with respective neutralizing antibodies before sponge implantation showed a significantly diminished angiogenic response (3.5%±0.5% in anti-VEGF-A treated, 3.2%±0.7% in anti-ANG treated, and 3.5%±0.6% in anti-VEGF-A/ANG treated). Further, both the normoxic and hypoxic ASC(CM) were able to sustain in vivo lymphangiogenesis in sponges. Collectively, the model demonstrated that the increased paracrine production of the VEGF-A and ANG in hypoxic-conditioned ASCs in vitro translated to an in vivo effect with a favorable biological significance. These results further illustrate the potential for utilization of an in vitro optimized ASC(CM) for in vivo angiogenesis-related applications as an effective cell-free technology.

An adipoinductive role of inflammation in adipose tissue engineering: key factors in the early development of engineered soft tissues.

Tissue engineering and cell implantation therapies are gaining popularity because of their potential to repair and regenerate tissues and organs. To investigate the role of inflammatory cytokines in new tissue development in engineered tissues, we have characterized the nature and timing of cell populations forming new adipose tissue in a mouse tissue engineering chamber (TEC) and characterized the gene and protein expression of cytokines in the newly developing tissues. EGFP-labeled bone marrow transplant mice and MacGreen mice were implanted with TEC for periods ranging from 0.5 days to 6 weeks. Tissues were collected at various time points and assessed for cytokine expression through ELISA and mRNA analysis or labeled for specific cell populations in the TEC. Macrophage-derived factors, such as monocyte chemotactic protein-1 (MCP-1), appear to induce adipogenesis by recruiting macrophages and bone marrow-derived precursor cells to the TEC at early time points, with a second wave of nonbone marrow-derived progenitors. Gene expression analysis suggests that TNFα, LCN-2, and Interleukin 1ß are important in early stages of neo-adipogenesis. Increasing platelet-derived growth factor and vascular endothelial cell growth factor expression at early time points correlates with preadipocyte proliferation and induction of angiogenesis. This study provides new information about key elements that are involved in early development of new adipose tissue.

Preparation of an adipogenic hydrogel from subcutaneous adipose tissue.

The ability to generate controlled amounts of adipose tissue would greatly ease the burden on hospitals for reconstructive surgery. We have previously shown that a tissue engineering chamber containing a vascular pedicle was capable of forming new fat; however, further refinements are required to enhance fat formation. The development and maintenance of engineered adipose tissue requires a suitable source of growth factors and a suitable scaffold. A hydrogel derived from adipose tissue may fulfil this need. Subcutaneous fat was processed into a thermosensitive hydrogel we refer to as adipose-derived matrix (ADM). Protein analysis revealed high levels of basement membrane proteins, collagens and detectable levels of growth factors. Adipose-derived stem cells exposed to this hydrogel differentiated into adipocytes with >90% efficiency and in vivo testing in rats showed significant signs of adipogenesis after 8 weeks. ADM's adipogenic properties combined with its simple gelation, relatively long shelf life and its tolerance to multiple freeze-thaw cycles, makes it a promising candidate for adipose engineering applications.

Long-term stability of adipose tissue generated from a vascularized pedicled fat flap inside a chamber.

BACKGROUND: Numerous studies demonstrate the generation and short-term survival of adipose tissue; however, long-term persistence remains elusive. This study evaluates long-term survival and transferability of de novo adipose constructs based on a ligated vascular pedicle and tissue engineering chamber combination. METHODS: Defined adipose tissue flaps were implanted into rats in either intact or perforated domed chambers. In half of the groups, the chambers were removed after 10 weeks and the constructs transferred on their vascular pedicle to a new site, where they were observed for a further 10 weeks. In the remaining groups, the tissue construct was observed for 20 weeks inside the chamber. Tissue volume was assessed using magnetic resonance imaging and histologic measures, and constructs were assessed for stability and necrosis. Sections were assessed histologically and for proliferation using Ki-67. RESULTS: At 20 weeks, volume analysis revealed an increase in adipose volume from 0.04 ± 0.001 ml at the time of insertion into the chambers to 0.27 ± 0.004 ml in the closed and 0.44 ± 0.014 ml in the perforated chambers. There was an additional increase of approximately 10 to 15 percent in tissue volume in flaps that remained in chambers for 20 weeks, whereas the volume of the transferred tissue not in chambers remained unaltered. Histomorphometric assessment of the tissues documented no signs of hypertrophy, fat necrosis, or atypical changes of the newly generated tissue. CONCLUSION: This study presents a promising new method of generating significant amounts of mature, vascularized, stable, and transferable adipose tissue for permanent autologous soft-tissue replacement.

Intrinsics and dynamics of fat grafts: an in vitro study.

BACKGROUND: Despite a revived interest in fat grafting procedures, clinicians still fail to demonstrate clearly the in vivo behavior of fat grafts as a dynamic tissue substitute. However, the basic principles in cellular biology teach us that cells can survive and develop, provided that a structural matrix exists that directs their behavior. The purpose of this in vitro study was to analyze that behavior of crude fat grafts, cultured on a three-dimensional laminin-rich matrix. METHODS: Nonprocessed, human fat biopsy specimens (approximately 1 mm) were inoculated on Matrigel-coated wells to which culture medium was added. The control group consisted of fat biopsy specimens embedded in medium alone. The cellular proliferation pattern was followed over 6 weeks. Additional cultures of primary generated cellular spheroids were performed and eventually subjected to adipogenic differentiation media. RESULTS: A progressive outgrowth of fibroblast-like cells from the core fat biopsy specimen was observed in both groups. Within the Matrigel group, an interconnecting three-dimensional network of spindle-shaped cells was established. This new cell colony reproduced spheroids that functioned again as solitary sources of cellular proliferation. Addition of differentiation media resulted in lipid droplet deposition in the majority of generated cells, indicating the initial steps of adipogenic differentiation. CONCLUSIONS: The authors noticed that crude, nonprocessed fat biopsy specimens do have considerable potential for future tissue engineering-based applications, provided that the basic principles of developmental, cellular biology are respected. Spontaneous in vitro expansion of the stromal cells present in fat grafts within autologous and injectable matrices could create "off-the-shelf" therapies for reconstructive procedures.

Epoxy-amine synthesised hydrogel scaffolds for soft-tissue engineering.

Highly porous and biodegradable hydrogels based on poly(ethylene glycol) (PEG) and cystamine (Cys) were fabricated using epoxy-amine chemistry and investigated as scaffolds for soft-tissue engineering. Whereas the application of fused-salt templates provided a comprehensive interconnecting pore morphology, the incorporation of a specially designed poly(epsilon-caprolactone) (PCL) cross-linker provided enhanced mechanical function without adversely effecting the scaffolds positive biological interactions. The addition of only 1.2 wt% of the PCL cross-linker was sufficient to provide improvements in the ultimate stress of 30-40%. In vitro studies not only confirmed the non-cytotoxic nature of the scaffolds, but also their degradation products, which were isolated and characterised by nuclear magnetic resonance (NMR) and matrix-assisted laser desorption/ionisation time-of-flight mass spectroscopy (MALDI ToF MS). In vivo trials were conducted over a period of 8 weeks through implantation of the scaffolds into the dorsal region of rats. At both 2 and 8 week time points the explants revealed complete infiltration by the surrounding tissue and the development of a vascular network to support the newly generated tissue, without an excessive foreign-body response.

Role of NADPH oxidase in tissue growth in a tissue engineering chamber in rats.

Previously we described a subcutaneous arteriovenous loop (AVL)-based tissue engineering chamber system, which contains an intrinsic circulation circuit created by joining the proximal ends of the femoral artery and vein with a venous graft. We showed that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was involved in mediating neovascularization inside the chamber. However, the role of NADPH oxidase in tissue formation in the chamber is unknown. In this study, we examined the effects of gp91ds-tat, a peptidyl inhibitor of NADPH oxidase, on the growth of engineered tissue blocks, using a rat chamber model. Chambers containing the AVL were filled with Matrigel mixed with gp91ds-tat (100 microM) or the scrambled control peptide. At 14 days, in control chambers, most of the Matrigel was replaced by granulation tissues; gp91ds-tat treatment significantly reduced the level of reactive oxygen species and retarded the tissue formation process. Although the total number of blood vessels per unit cellularized area was not different between two groups, most vessels in gp91ds-tat-treated tissues had smaller lumens as compared to control. The total area occupied by vessel lumens was much less in gp91ds-tat-treated tissues (10.3 +/- 1.3% in control vs. 1.7 +/- 0.5% in gp91ds-tat group; p < 0.001). In vitro, gp91ds-tat treatment reduced proliferation and migration of cultured microvascular endothelial cells. Our data suggest that inhibition of NADPH oxidase function retards tissue formation in the tissue engineering chamber, which may be related to the malformed new blood vessels in the engineered tissue.

Adipose differentiation of bone marrow-derived mesenchymal stem cells using Pluronic F-127 hydrogel in vitro.

Due to increasing clinical demand for adipose tissue, a suitable scaffold for engineering adipose tissue constructs is needed. In this study, we have developed a three-dimensional (3-D) culture system using bone marrow-derived mesenchymal stem cells (BM-MSC) and a Pluronic F-127 hydrogel scaffold as a step towards the in vitro tissue engineering of fat. BM-MSC were dispersed into a Pluronic F-127 hydrogel with or without type I collagen added. The adipogenic differentiation of the BM-MSC was assessed by cellular morphology and further confirmed by Oil Red O staining. The BM-MSC differentiated into adipocytes in Pluronic F-127 in the presence of adipogenic stimuli over a period of 2 weeks, with some differentiation present even in absence of such stimuli. The addition of type I collagen to the Pluronic F-127 caused the BM-MSC to aggregate into clumps, thereby generating an uneven adipogenic response, which was not desirable.

Spontaneous large volume adipose tissue generation from a vascularized pedicled fat flap inside a chamber space.

A novel method of spontaneous generation of new adipose tissue from an existing fat flap is described. A defined volume of fat flap based on the superficial inferior epigastric vascular pedicle in the rat was elevated and inset into a hollow plastic chamber implanted subcutaneously in the groin of the rat. The chamber walls were either perforated or solid and the chambers either contained poly(D,L-lactic-co-glycolic acid) (PLGA) sponge matrix or not. The contents were analyzed after being in situ for 6 weeks. The total volume of the flap tissue in all groups except the control groups, where the flap was not inserted into the chambers, increased significantly, especially in the perforated chambers (0.08 +/- 0.007 mL baseline compared to 1.2 +/- 0.08 mL in the intact ones). Volume analysis of individual component tissues within the flaps revealed that the adipocyte volume increased and was at a maximum in the chambers without PLGA, where it expanded from 0.04 +/- 0.003 mL at insertion to 0.5 +/- 0.08 mL (1250% increase) in the perforated chambers and to 0.16 +/- 0.03 mL (400% increase) in the intact chambers. Addition of PLGA scaffolds resulted in less fat growth. Histomorphometric analysis rather than simple hypertrophy documented an increased number of adipocytes. The new tissue was highly vascularized and no fat necrosis or atypical changes were observed.

Monocyte chemoattractant protein-1 and nitric oxide promote adipogenesis in a model that mimics obesity.

OBJECTIVE: An increasing body of evidence is emerging linking adipogenesis and inflammation. Obesity, alone or as a part of the metabolic syndrome, is characterized by a state of chronic low-level inflammation as revealed by raised plasma levels of inflammatory cytokines and acute-phase proteins. If inflammation can, in turn, increase adipose tissue growth, this may be the basis for a positive feedback loop in obesity. We have developed a tissue engineering model for growing adipose tissue in the mouse that allows quantification of increases in adipogenesis. In this study, we evaluated the adipogenic potential of the inflammogens monocyte chemoattractant protein (MCP)-1 and zymosan-A (Zy) in a murine tissue engineering model. RESEARCH METHODS AND PROCEDURES: MCP-1 and Zy were added to chambers filled with Matrigel and fibroblast growth factor 2. To analyze the role of inducible nitric oxide synthase (iNOS), the iNOS inhibitor aminoguanidine was added to the chamber. RESULTS: Our results show that MCP-1 generated proportionally large quantities of new adipose tissue. This neoadipogenesis was accompanied by an ingrowth of macrophages and could be mimicked by Zy. Aminoguanidine significantly inhibited the formation of adipose tissue. DISCUSSION: Our findings demonstrate that low-grade inflammation and iNOS expression are important factors in adipogenesis. Because fat neoformation in obesity and the metabolic syndrome is believed to be mediated by macrophage-derived proinflammatory cytokines, this adipose tissue engineering system provides a model that could potentially be used to further unravel the pathogenesis of these two metabolic disorders.

Adipose tissue engineering based on the controlled release of fibroblast growth factor-2 in a collagen matrix.

Adipose tissue forms when basement membrane extract (Matrigel) and fibroblast growth factor-2 (FGF-2) are added to our mouse tissue engineering chamber model. A mouse tumor extract, Matrigel is unsuitable for human clinical application, and finding an alternative to Matrigel is essential. In this study we generated adipose tissue in the chamber model without using Matrigel by controlled release of FGF-2 in a type I collagen matrix. FGF-2 was impregnated into biodegradable gelatin microspheres for its slow release. The chambers were filled with these microspheres suspended in 60 microL collagen gel. Injection of collagen containing free FGF-2 or collagen containing gelatin microspheres with buffer alone served as controls. When chambers were harvested 6 weeks after implantation, the volume and weight of the tissue obtained were higher in the group that received collagen and FGF-2 impregnated microspheres than in controls. Histologic analysis of tissue constructs showed the formation of de novo adipose tissue accompanied by angiogenesis. In contrast, control groups did not show extensive adipose tissue formation. In conclusion, this study has shown that de novo formation of adipose tissue can be achieved through controlled release of FGF-2 in collagen type I in the absence of Matrigel.

Generation of a vascularized organoid using skeletal muscle as the inductive source.

The technology required for creating an in vivo microenvironment and a neovasculature that can grow with and service new tissue is lacking, precluding the possibility of engineering complex three-dimensional organs. We have shown that when an arterio-venous (AV) loop is constructed in vivo in the rat groin, and placed inside a semisealed chamber, an extensive functional vasculature is generated. To test whether this unusually angiogenic environment supports the survival and growth of implanted tissue or cells, we inserted various preparations of rat and human skeletal muscle. We show that after 6 weeks incubation of muscle tissue, the chamber filled with predominantly well-vascularized recipient-derived adipose tissue, but some new donor-derived skeletal muscle and connective tissue were also evident. When primary cultured myoblasts were inserted into the chamber with the AV loop, they converted to mature striated muscle fibers. Furthermore, we identify novel adipogenesis-inducing properties of skeletal muscle. This represents the first report of a specific three-dimensional tissue grown on its own vascular supply.

Endometrial arteriogenesis: vascular smooth muscle cell proliferation and differentiation during the menstrual cycle and changes associated with endometrial bleeding disorders.

The cyclical regrowth of the human endometrial vasculature every 28 days following menstruation provides an excellent opportunity for studying arteriogenesis in a human tissue. Despite this, very little has been published to date about temporal or spatial changes in endometrial arteriolar structure or function. Immunohistochemical studies using vascular smooth muscle cell (VSMC) differentiation markers have identified spatially organised differences in VSMC phenotype. However, these have not significantly increased our understanding of how or when arteriogenesis occurs. Endometrial VSMC proliferation continues at a steady rate throughout the cycle, increasing in the specialised spiral arterioles in the mid and late secretory phases. Although estrogen and progesterone play a dominant role in regulating endometrial growth and regression, their direct involvement in arteriogenesis is less clear. Estrogen and progesterone receptors have been reported in endometrial VSMC, although heterogeneity of expression occurs. The angiopoietin/Tie axis has been investigated in endometrium, although the results of 3 studies published to date are contradictory, making conclusions about a role in arteriogenesis problematic. Abnormalities of arteriogenesis may play a role in disorders such as menorrhagia and breakthrough bleeding in contraceptive and hormone replacement therapy users. There is evidence that VSMC proliferation is reduced in spiral arterioles of women with menorrhagia, along with reduced pulsatility index of the uterine artery as shown by Doppler ultrasound. In women taking progestin-only contraception, endometrial perivascular alpha smooth muscle actin is reduced in those who suffer from breakthrough bleeding compared to those that don't. Considerable work is still required to elucidate when in the cycle, where in the tissue, and what mechanisms regulate endometrial arteriogenesis.