Mature Vessel Density Research Articles

Equine ovarian tissue xenografting: impacts of cooling, vitrification, and VEGF.

Ovarian tissue transplantation methods using cooled and cryopreserved samples have been attractive options for fertility preservation in animal models and humans. The aim of this study was to evaluate the impact of previous exposure to cooling, cryopreservation, and VEGF on the overall efficiency of equine ovarian tissue after heterotopic xenotransplantation in mice. The end points evaluated were follicular morphology and development, follicular and stromal cell densities, angiogenesis (i.e. the density of new and mature blood vessels), collagen types I and III fiber densities, and total fibrosis. Ovaries of adult mares were harvested after ovariectomy, and ovarian fragments were xenografted in the i.p. wall of BALB nude mice. Ten types of treatments involving different combinations of cooling, cryopreservation, xenografting procedures, and VEGF exposure were compared. The novel aspect of this study was the use of equine ovarian tissue xenotransplantation in mice, challenging the fragments with different combinations of treatments. The main findings were (i) cooling but not cryopreservation was effective in preserving the follicular morphology, (ii) a greater percentage of developing follicles but lower follicular and stromal cell densities were observed after ovarian tissue engraftment, (iii) exposure to VEGF increased new and mature vessels in cryopreserved-transplanted tissue, and (iv) an appropriate balance in the collagen types I and III fiber ratio in cooling-transplanted tissue was observed after exposure to VEGF. This study contributes to advancing knowledge in the preservation of ovarian tissue after cooling-cryopreservation and transplantation aiming to be applied to genetically superior/valuable horses, livestock, endangered animals, and, possibly, humans.Lay summaryDue to ethical limitations involving humans, the female horse (mare) has recently emerged as an alternative model for reproductive comparisons with women to optimize fertility restoration using ovarian tissue transplantation techniques. This study determined if ovarian tissue from donor mares (n = 3), exposed or not to vascular endothelial growth factor (VEGF) before transplantation, better survives for 7 days after transplantation into mouse hosts (n = 12). Tissues submitted to different combinations of cooling, freezing, and transplanting treatments, along with control groups, were evaluated using the parameters morphology, development, the density of immature eggs (follicles), the density of supportive (stromal) cells, collagen protein proportions, and density of blood vessels. Frozen-thawed treatments had lower percentages of normal follicles. Exposure to VEGF increased blood vessel densities in frozen tissue and favored adequate collagen levels in cooled-transplanted treatments. In conclusion, VEGF exposure seems to be beneficial for mare ovarian tissue transplantation and warrants further investigation.

The neovascularization effect of dedifferentiated fat cells

Mature adipocyte-derived dedifferentiated fat (DFAT) cells can be prepared efficiently and with minimal invasiveness to the donor. They can be utilized as a source of transplanted cells during therapy. Although the transplantation of DFAT cells into an ischemic tissue enhances angiogenesis and increases vascular flow, there is little information regarding the mechanism of the therapeutic angiogenesis. To further study this, mice ischemic hindlimb model was used. It was confirmed that in comparison with the adipose derived stem cells and fibroblasts, the transplantation of DFAT cells led to a significant improvement in the blood flow and increased mature blood vessel density. The ability of DFAT cells to secrete angiogenic factors in hypoxic conditions and upon co-culture with vascular endothelial cells was then examined. Furthermore, we examined the possibility that DFAT cells differentiating into pericytes. The therapeutic angiogenic effects of DFAT cells were observed by the secretion of angiogenic factors and pericyte differentiation by transforming growth factor β1 signalling via Smad2/3. DFAT cells can be prepared with minimal invasiveness and high efficiency and are expected to become a source of transplanted cells in the future of angiogenic cell therapy.

Layered smooth muscle cell–endothelial progenitor cell sheets derived from the bone marrow augment postinfarction ventricular function

ObjectiveThe angiogenic potential of endothelial progenitor cells (EPCs) may be limited by the absence of their natural biologic foundation, namely smooth muscle pericytes. We hypothesized that joint delivery of EPCs and smooth muscle cells (SMCs) in a novel, totally bone marrow–derived cell sheet will mimic the native architecture of a mature blood vessel and act as an angiogenic construct to limit post infarction ventricular remodeling. MethodsPrimary EPCs and mesenchymal stem cells were isolated from bone marrow of Wistar rats. Mesenchymal stem cells were transdifferentiated into SMCs by culture on fibronectin-coated culture dishes. Confluent SMCs topped with confluent EPCs were detached from an Upcell dish to create a SMC-EPC bi-level cell sheet. A rodent model of ischemic cardiomyopathy was then created by ligating the left anterior descending artery. Rats were randomized into 3 groups: cell sheet transplantation (n = 9), no treatment (n = 12), or sham surgery control (n = 7). ResultsFour weeks postinfarction, mature vessel density tended to increase in cell sheet-treated animals compared with controls. Cell sheet therapy significantly attenuated the extent of cardiac fibrosis compared with that of the untreated group (untreated vs cell sheet, 198 degrees [interquartile range (IQR), 151-246 degrees] vs 103 degrees [IQR, 92-113 degrees], P = .04). Furthermore, EPC-SMC cell sheet transplantation attenuated myocardial dysfunction, as evidenced by an increase in left ventricular ejection fraction (untreated vs cell sheet vs sham, 33.5% [IQR, 27.8%-35.7%] vs 45.9% [IQR, 43.6%-48.4%] vs 59.3% [IQR, 58.8%-63.5%], P = .001) and decreases in left ventricular dimensions. ConclusionsThe bone marrow-derived, spatially arranged SMC-EPC bi-level cell sheet is a novel, multilineage cellular therapy obtained from a translationally practical source. Interactions between SMCs and EPCs augment mature neovascularization, limit adverse remodeling, and improve ventricular function after myocardial infarction.

Cardiosphere-Derived Cells Require Endoglin for Paracrine-Mediated Angiogenesis.

SummaryClinical trials of stem cell therapy to treat ischemic heart disease primarily use heterogeneous stem cell populations. Small benefits occur via paracrine mechanisms that include stimulating angiogenesis, and increased understanding of these mechanisms would help to improve patient outcomes. Cardiosphere-derived-cells (CDCs) are an example of these heterogeneous stem cell populations, cultured from cardiac tissue. CDCs express endoglin, a co-receptor that binds specific transforming growth factor β (TGFβ) family ligands, including bone morphogenetic protein 9 (BMP9). In endothelial cells endoglin regulates angiogenic responses, and we therefore hypothesized that endoglin is required to promote the paracrine pro-angiogenic properties of CDCs. Cre/LoxP technology was used to genetically manipulate endoglin expression in CDCs, and we found that the pro-angiogenic properties of the CDC secretome are endoglin dependent both in vitro and in vivo. Importantly, BMP9 pre-treatment of endoglin-depleted CDCs restores their pro-angiogenic paracrine properties. As BMP9 signaling is normally required to maintain endoglin expression, we propose that media containing BMP9 could be critical for therapeutic CDC preparation.

Autologous cell therapy with CD133+ bone marrow-derived stem cells for refractory Asherman's syndrome and endometrial atrophy: a pilot cohort study.

Could cell therapy using autologous peripheral blood CD133+ bone marrow-derived stem cells (BMDSCs) offer a safe and efficient therapeutic approach for patients with refractory Asherman's syndrome (AS) and/or endometrial atrophy (EA) and a wish to conceive? In the first 3 months, autologous cell therapy, using CD133+ BMDSCs in conjunction with hormonal replacement therapy, increased the volume and duration of menses as well as the thickness and angiogenesis processes of the endometrium while decreasing intrauterine adhesion scores. AS is characterized by the presence of intrauterine adhesions and EA prevents the endometrium from growing thicker than 5 mm, resulting in menstruation disorders and infertility. Many therapies have been attempted for these conditions, but none have proved effective. This was a prospective, experimental, non-controlled study. There were 18 patients aged 30-45 years with refractory AS or EA were recruited, and 16 of these completed the study. Medical history, physical examination, endometrial thickness, intrauterine adhesion score and neoangiogenesis were assessed before and 3 and 6 months after cell therapy. After the initial hysteroscopic diagnosis, BMDSC mobilization was performed by granulocyte-CSF injection, then CD133+ cells were isolated through peripheral blood aphaeresis to obtain a mean of 124.39 million cells (range 42-236), which were immediately delivered into the spiral arterioles by catheterization. Subsequently, endometrial treatment after stem cell therapy was assessed in terms of restoration of menses, endometrial thickness (by vaginal ultrasound), adhesion score (by hysteroscopy), neoangiogenesis and ongoing pregnancy rate. The study was conducted at Hospital Clínico Universitario of Valencia and IVI Valencia (Spain). All 11 AS patients exhibited an improved uterine cavity 2 months after stem cell therapy. Endometrial thickness increased from an average of 4.3 mm (range 2.7-5) to 6.7 mm (range 3.1-12) ( ITALIC! P = 0.004). Similarly, four of the five EA patients experienced an improved endometrial cavity, and endometrial thickness increased from 4.2 mm (range 2.7-5) to 5.7 mm (range 5-12) ( ITALIC! P = 0.03). The beneficial effects of the cell therapy increased the mature vessel density and the duration and intensity of menses in the first 3 months, with a return to the initial levels 6 months after the treatment. Three patients became pregnant spontaneously, resulting in one baby boy born, one ongoing pregnancy and a miscarriage. Furthermore, seven pregnancies were obtained after fourteen embryo transfers, resulting in three biochemical pregnancies, one miscarriage, one ectopic pregnancy, one baby born and one ongoing pregnancy. Limitations of this pilot study include the small sample size and the lack of control group. This novel autologous cell therapy is a promising therapeutic option for patients with these incurable pathologies and a wish to conceive. This study was funded by the Spanish Ministry of Science and Innovation (SAF 2012-31017, Principal Investigator C.S.), Spanish Ministry of Health (EC11-299, Principal Investigator C.S.) and Regional Valencian Ministry of Education (PROMETEOII/2013/018, Principal Investigator C.S.). Four authors (X.S., I.C., A.P. and C.S.) are co-inventors of the patent resulting from this work (Application number: 62/013,121). S.C., C.A., F.R., J.F., J.P. and J.R. have no conflict of interest in relation to this work. This study was registered with ClinicalTrials.gov (NCT02144987).

Cystathionine γ-lyase regulates arteriogenesis through NO-dependent monocyte recruitment.

Hydrogen sulfide (H2S) is a vasoactive gasotransmitter that is endogenously produced in the vasculature by the enzyme cystathionine γ-lyase (CSE). However, the importance of CSE activity and local H2S generation for ischaemic vascular remodelling remains completely unknown. In this study, we examine the hypothesis that CSE critically regulates ischaemic vascular remodelling involving H2S-dependent mononuclear cell regulation of arteriogenesis. Arteriogenesis including mature vessel density, collateral formation, blood flow, and SPY angiographic blush rate were determined in wild-type (WT) and CSE knockout (KO) mice at different time points following femoral artery ligation (FAL). The role of endogenous H2S in regulation of IL-16 expression and subsequent recruitment of monocytes, and expression of VEGF and bFGF in ischaemic tissues, were determined along with endothelial progenitor cell (CD34/Flk1) formation and function. FAL of WT mice significantly increased CSE activity, expression and endogenous H2S generation in ischaemic tissues, and monocyte infiltration, which was absent in CSE-deficient mice. Treatment of CSE KO mice with the polysulfide donor diallyl trisulfide restored ischaemic vascular remodelling, monocyte infiltration, and cytokine expression. Importantly, exogenous H2S therapy restored nitric oxide (NO) bioavailability in CSE KO mice that was responsible for monocyte recruitment and arteriogenesis. Endogenous CSE/H2S regulates ischaemic vascular remodelling mediated during hind limb ischaemia through NO-dependent monocyte recruitment and cytokine induction revealing a previously unknown mechanism of arteriogenesis.

A rabbit model of fat graft recipient site preconditioning using external negative pressure.

BackgroundFat is widely used in soft tissue augmentation. Nevertheless, it has an unpredictably high resorption rate. Clinically, external expansion with negative pressure is used to increase fat graft survival. In this study, fat graft recipient sites were preconditioned by external application of negative pressure in order to test for improvements in vascularity and fat graft survival.MethodsNegative pressure was applied randomly to either the left or right dorsal ear of 20 New Zealand male white rabbits at a pressure of -125 mm Hg. The negative pressure was removed one week after the skin perfusion was measured. The skin flap at each ear was elevated, and 1 g of fat was grafted above the dorsal perichondrium. After one week, the fat weight, microvessel density, mature vessel density of the skin and fat, and amount of glycerol released were measured. Three months after the grafting, the same measurements were performed, with the exception of glycerol release.ResultsThe fat survival rate of the experimental group (75.4%±3.9%) was higher than that of the control group (53.1%±4.3%) (P<0.001). Skin perfusion was higher in the experimental group. The glycerol release in the experimental group was significantly higher than in the control. The microvessel density of the skin and fat was significantly higher in the experimental group. Three months after the grafting, the skin and fat mature vessel density was significantly higher in the experimental groups.ConclusionsNegative pressure prior to fat grafting increased the vascularity of the recipient site, and, accordingly, enhanced fat graft survival.

P06 CSE/H2S rescues impaired arteriogenesis in mouse hind limb ischemia via IL-16 dependant monocyte recruitment and expression of bFGF

Although animal studies concerning cytokines or stem cell therapies have shown hope for arteriogenesis, clinical results have been disappointing due to multi-factoral causes. Monocyte signaling is a dominant and important mediator of arteriogenesis activity and monocyte-signaling pathways can be severely impaired during various cardiovascular disorders. Hydrogen sulfide (H 2 S) is produced by cystathionine- γ lyase (CSE), and a potential gaseous molecule that has many positive cytoprotective effects including regulation of monocyte functions. However, a molecular mechanism of CSE/H2S regulation of arteriogenesis during ischemia is not established. Therefore, we hypothesized that CSE/H 2 S modulates arterial collateral formation in mouse hind limb ischemia via IL-16/bFGF dependent pathway. Unilateral permanent hind limb ischemia was induced in wild type (WT) and CSE −/− mice. Mice were then randomly assigned into four groups (8 mice, each group); WT PBS, WT DATS (a hydrogen sulfide donor, 200 μ g/kg, twice daily, IV until day 21), CSE - PBS, CSE −/− DATS. CSE activity and H 2 S levels in monocytes and tissues were determined in ischemic conditions and with inflammatory agents and studied both in vitro and in vivo. Tissue perfusion was determined using laser Doppler perfusion imaging system and the number and diameter of arterial collaterals measured using SPY angiography. Mature vessel density and monocyte recruitment was investigated by α -SMA/CD31/DAPI and MAC-2/DAPI positive staining. ELISA was used to measure IL-16 and bFGF levels. CSE activity and free H 2 S levels were increased during ischemia and TNF- α stimulation in cultured monocytes. CSE activity and H 2 S levels were also elevated in bone marrow derived and circulating monocytes, and tissues after ischemia induction in WT mice but not from CSE −/− mice. Blood perfusion, blush rate, number and diameter of collaterals, and mature vessel density were decreased in CSE −/− mice compared to WT mice that was rescued by DATS therapy. IL-16 levels were reduced in ischemic tissue of CSE −/− mice compared to WT mice. IL-16 levels were restored by DATS treatment in ischemic tissue of CSE - mice. Monocyte recruitment was significantly reduced in ischemic tissues of CSE −/− mice that were rescued by DATS treatment. Lastly bFGF levels were less in ischemic tissue of CSE −/− mice compared to WT mice and that were restored by DATS therapy. CSE/H 2 S regulates arteriogenesis via IL-16 mediated monocyte recruitment and up-regulation of bFGF levels in ischemic tissue of CSE −/− mice. These findings reveal a novel role of monocyte dependent CSE/H 2 S in regulating arteriogenesis activity.

Promoted regeneration of mature blood vessels by electrospun fibers with loaded multiple pDNA-calcium phosphate nanoparticles

Vascularization is one of the capital challenges in the establishment of tissue engineering constructs and recovery of ischemic and wounded tissues. The aim of this study was to assess electrospun fibers with loadings of multiple pDNA to allow a localized delivery for an efficient regeneration of mature blood vessels. To induce sufficient protein expression, a reverse microemulsion process was adopted to load pDNA into calcium phosphate nanoparticles (CP-pDNA), which were electrospun into fibers to achieve a sustained release for 4weeks. Compared with pDNA-infiltrated fibers, the localized and gradual release of pDNA facilitated cell proliferation, gene transfection, and extracellular matrix secretion and enhanced the generation of blood vessels after subcutaneous implantation. Compared with commonly used pDNA polyplexes with poly(ethyleneimine), CP-pDNA nanoparticles induced significantly lower cytotoxicity and less inflammation reaction after implantation into animals. Fibers with encapsulated nanoparticles containing plasmids encoding vascular endothelial growth factor (pVEGF) and basic fibroblast growth factors (pbFGF) led to significantly higher density of mature blood vessels than those containing individual plasmid. It is suggested that the integration of CP-pDNA nanoparticles with loadings of multiple plasmids into fibrous scaffolds should provide clinical relevance for therapeutic vascularization, getting fully vascularized in engineered tissues and regeneration of blood vessel substitutes.

The Effect of Platelet-Rich Plasma on Flap Survival in Random Extension of an Axial Pattern Flap in Rabbits

Autologous platelet-rich plasma contains multiple growth factors. The aim of this randomized trial was to study the effect of an injection of platelet-rich plasma during random extension of an axial pattern flap procedure. Sixteen male New Zealand white rabbits were assigned randomly to two groups of eight animals each. A 10 × 10-cm unipedicled fasciocutaneous island flap was elevated. After flap elevation, the experimental group was injected evenly with platelet-rich plasma. For the control group, normal saline was injected into the same areas. The flap survival area was assessed 2 weeks after the operation. Microvessel density and mature vessel density were evaluated to examine the angiogenic and arteriogenic effects, respectively. Flap survival was significantly increased in the experimental group (80.3 ± 10.0 percent) compared with the control group (55.2 ± 13.6 percent; p = 0.001). Immunohistologic staining showed that the microvessel density (38.8 ± 11.0 per high-power field for the experimental group; 17.6 ± 6.6 per high-power field for the control group; p = 0.001) and mature vessel density (16.3 ± 4.1 per high-power field for the experimental group; 7.4 ± 1.7 per high-power field for the control group; p = 0.001) were significantly higher in the experimental group than in the control group. The authors' data indicate that platelet-rich plasma injection significantly improves the surviving area of flaps through angiogenic and arteriogenic effects. Thus, it could be used in a clinical situation as a minimally invasive, fast, and easily applicable treatment.

Creating 3D Angiogenic Growth Factor Gradients in Fibrous Constructs to Guide Fast Angiogenesis

Fast angiogenesis in 3D fibrous constructs that mimic the morphology of the extracellular matrix remains challenging due to limited porosity in the densely packed constructs. We investigated whether mimicking the in vivo chemotaxis microenvironment for native blood vessel formation would stimulate angiogenesis in the fibrous constructs. The chemotaxis microenvironment was created by introducing 3D angiogenic growth factor gradients into the constructs. We have developed a technique that can quickly fabricate (∼40 min) such 3D gradients by simultaneously electrospinning polycaprolactone (PCL) fibers, encapsulating gradient amount of bFGF (stabilized by heparin) into poly(lactide-co-glycolide) (PLGA) microspheres, and electrospraying the microspheres into PCL fibers. Gradient formation was confirmed by fluorescence microscopy. Gradients with different steepnesses were obtained by modulating the initial concentration of the bFGF solution. All of the constructs were able to sustainedly release bioactive bFGF over a 28 day period. The release kinetics was dependent on the bFGF loading and steepness of the gradient. In vitro cell migration study demonstrated that bFGF gradients significantly increased the depth of cell migration. To assess the efficacy of bFGF gradients in inducing angiogenesis, we implanted constructs subcutaneously using mouse model. bFGF gradients significantly promoted cell penetration into the constructs. After 10 days of implantation, a high density of mature blood vessels (positive to both CD31 and α-SMA) were formed in the constructs. Vessel density was increased with the increase in steepness of the bFGF gradient. These gradient constructs may have potential to engineer vascularized tissues for various applications.

P61 Hydrogen sulfide therapy rescues critical limb ischemia in aged diabetic mice through NO/VEGF dependent pathway

Hydrogen sulfide is a gaseous signaling molecule which is produced in vivo by three enzymes such as cystathionine γ-lyase (CSE), cystathionine β- synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MST) from L-cysteine.CSE is the prime enzyme among others for sulfide production in vascular tissues of our body. Hydrogen sulfide is reported to have vasodilatory, anti–inflammatory, anti-oxidant, cytoprotective and pro-angiogenic effects. Sulfide production is decreased in diabetic condition. However, the molecular mechanisms of sulfide-mediated protection during chronic tissue ischemia in diabetic condition are completely unknown. To determine molecular mechanisms involved in hydrogen sulfide mediated cytoprotection during chronic tissue ischemia in diabetic condition. Hind limb ischemia was induced in 10 week old wild type, 42 week old Db/Db diabetic or 10–12 week old CSE knock out (CSEKO, n = 8, each group). PBS, 0.1, 0.5 and 1 mg/kg sodium sulfide (Na 2 S) was administered twice daily by retro-orbital injection. Hind limb perfusion was measured using a laser Doppler perfusion probe. SPY imaging was performed to determine the collateral formation in different time points. Capillary myofiber ratio was determined by NOVA Red staining with CD31 antibody. Mature vessel density and proliferation index were determined by the ratio of smooth muscle actin (SMA) to DAPI and Ki67 to DAPI positive areas respectively. TUNEL assay was measured to investigate the apoptosis. Nitric oxide (NO) was measured by NO analyzer (NOA). CPTIO (1 mg/kg/day) was injected intra-peritoneally to determine the effect of NO in sulfide induced ischemic angiogenesis. VEGF aptamer (50 mg/kg/day) was injected locally to the ischemic muscle to determine the role of VEGF in sulfide induced ischemic angiogenesis. Lastly, VEGF expression was determined by ELISA. Blood perfusion, blush rate by SPY imaging, angiogenic index, proliferation index and mature vessel density were significantly decreased in CSEKO mice compared WT mice indicating the role of sulfide on ischemic angiogeneis and arteriogenesis. Na 2 S dose dependently increases the angiogenesis index, proliferation index, mature vessel density and restores the blood flow in WT mice. Hydrogen sulfide also rescues the impaired blood flow in CSEKO mice by increasing angiogenic index, proliferation index and mature vessel density. Blood tissue perfusion, proliferation index, capillary to myofiber ratio and VEGF expression were all significantly increased and conversely, apoptosis was prevented in ischemic hind limbs of aged diabetic mice treated with Na 2 S compared to PBS control. There was no effect of sulfide on blood glucose, body weight and lipid profiles of diabetic mice.Total NO production was increased in sulfide treated group compared to PBS which was inhibited by CPTIO treatment. Lastly, VEGF164 aptamer blocked sulfide induced augmentation of blood flow and reduced the expression of VEGF164 in WT and diabetic mice hind limb ischemia indicating VEGF164 as the sulfide induced angiogenic stimulator. Sodium sulfide therapy restores tissue perfusion of critical limb ischemia by increasing production of NO, increasing expression of VEGF and preventing apoptosis in diabetic mouse chronic hind limb ischemia which may be beneficial for the diabetic patient with peripheral vascular disease.

Microfabrication of proangiogenic cell-Laden alginate-g-Pyrrole hydrogels

Cells have been extensively studied for their uses in various therapies because of their capacities to produce therapeutic proteins and recreate new tissues. It has often been suggested that the efficacy of cell therapies can greatly be improved through the ability to localize and regulate cellular activities at a transplantation site; however, the technologies for this control are lacking. Therefore, this study reports a cell-Laden hydrogel patch engineered to support the proliferation and angiogenic growth factor expression of cells adhered to their surfaces, and to further promote neovascularization. Hydrogels consisting of alginate chemically linked with pyrrole units, termed alginate-g-pyrrole, were prepared through an oxidative cross-linking reaction between pyrrole units. Fibroblasts adhered to the alginate-g-pyrrole hydrogels, and exhibited increased proliferation and overall vascular endothelial growth factor (VEGF) expression, compared to those on pyrrole-free hydrogels. Furthermore, the alginate-g-pyrrole hydrogel surfaces were modified to present microposts, subsequently increasing the amount of pyrrole units on their surfaces. Cells adhered to the microfabricated gel surfaces exhibited increased proliferation and overall VEGF expression proportional to the density of the microposts. The resulting micropatterned alginate-g-pyrrole hydrogels exhibited increases in the size and density of mature blood vessels when implanted on chick chorioallantoic membranes (CAMs). The hydrogel system developed in this study will be broadly useful for improving the efficacy of a wide array of cell-based wound healing and tissue regenerative therapies.

Controlled release of thymosin β4 from injected collagen-chitosan hydrogels promotes angiogenesis and prevents tissue loss after myocardial infarction.

Acute myocardial infarction (MI) leads to fibrosis and severe left ventricular wall thinning. Enhancing vascularization within the infarct reduces cell death and maintains a thick left ventricular wall, which is essential for proper cardiac function. Here, we evaluated the controlled delivery of thymosin β4 (Tβ4), which supports cardiomyocyte survival by inducing vascularization and upregulating Akt activity, in the treatment of MI. We injected collagen-chitosan hydrogel with controlled release of Tβ4 into the infarct after performing left anterior descending artery ligation in rats. Tβ4-encapsulated hydrogel (thymosin) significantly reduced tissue loss post-MI (13 ± 4%), compared with 58 ± 3% and 30 ± 8% tissue loss for no treatment (MI only) and Tβ4-free hydrogel (control). Significantly more Factor VIII-positive blood vessels with diameter >50 µm were in the thymosin group compared with both MI only and control (p < 0.0001), showing Tβ4-induced vascularization. Wall thickness was positively correlated with the mature blood vessel density (r = 0.9319; p < 0.0001). Controlled release of Tβ4 within the infarct enhances angiogenesis and presence of cardiomyocytes that are necessary for cardiac repair.

Multiple release of polyplexes of plasmids VEGF and bFGF from electrospun fibrous scaffolds towards regeneration of mature blood vessels

Key challenges associated with the outcomes of vascular grafting (for example, to fully vascularize engineered tissues and promptly regenerate blood vessel substitutes) remain unsolved. The local availability of angiogenic growth factors is highly desirable for tissue regeneration, and plasmid loading in scaffolds represents a powerful alternative for local production of tissue-inductive factors. No attempt has been made so far to clarify the efficacy of electrospun fibers with the loading of multiple plasmids to promote tissue regeneration. In the present study, core–sheath electrospun fibers with the encapsulation of polyplexes of basic fibroblast growth factor-encoding plasmid (pbFGF) and vascular endothelial growth factor-encoding plasmid (pVEGF) were evaluated to promote the generation of mature blood vessels. In vitro release indicated a sustained release of pDNA for ∼4weeks with as low as ∼10% initial burst release, and the release patterns from the single and twofold plasmid-loaded systems coincided. In vitro investigations on human umbilical vein endothelial cells showed that the sustained release of pDNA from fibrous mats promoted cell attachment and viability, cell transfection and protein expression, and extracellular secretion of collagen IV and laminin. The acceleration of angiogenesis was assessed in vivo after subcutaneous implantation of fibrous scaffolds, and the explants were evaluated after 1, 2 and 4weeks’ treatment by histological and immunohistochemical staining. Compared with pDNA polyplex infiltrated fibrous mats, the pDNA polyplex encapsulated fibers alleviated the inflammation reaction and enhanced the generation of microvessels. Although there was no significant difference in the total number of microvessels, the density of mature vessels was significantly enhanced by the combined treatment with both pbFGF and pVEGF compared with those incorporating individual pDNA. The integration of the core–sheath structure, DNA condensation and multiple delivery strategies provided a potential platform for scaffold fabrication to regenerate functional tissues.

Therapeutic Treatment with Sustained-Release Platelet-Rich Plasma Restores Blood Perfusion by Augmenting Ischemia-Induced Angiogenesis and Arteriogenesis in Diabetic Mice

Objective: The objective of this investigation was to establish the effectiveness of sustained-release platelet-rich plasma (PRP) on perfusion and neovascularization in diabetic murine hind limb ischemia. Methods: After surgery in streptozotocin-induced diabetic mice, the mice were randomly assigned to the following 4 experimental groups: control (C), 100 µl of the sustained-release form of platelet-poor plasma (PPP), 100 µl of the solution form of PRP (PRP-sol), and 100 µl of the sustained-release form of PRP (PRP-sr). Endpoint evaluations were: blood perfusion by laser Doppler perfusion imaging (LDPI), vascular density by anti-vWF, and mature vessel density by anti-smooth muscle actin antibody. Results: This study demonstrated that a sustained release of PRP increases the perfusion of ischemic tissue as measured by LDPI (57 ± 12; 56 ± 9; 72 ± 7, and 98 ± 4 for the C, PPP, PRP-sol, and PRP-sr groups, respectively; p < 0.05), capillary density (151 ± 16; 158 ± 12; 189 ± 39, and 276 ± 39 for groups C, PPP, PRP-sol, and PRP-sr, respectively; p < 0.05), and mature vessel density (28 ± 2; 31 ± 3; 52 ± 10, and 85 ± 13 for the C, PPP, PRP-sol, and PRP-sr groups, respectively; p < 0.05). Conclusion: A sustained release of PRP containing potent angiogenic growth factors restores blood perfusion by stimulating angiogenesis and arteriogenesis.

Therapeutic Angiogenesis by Transplantation of Human Embryonic Stem Cell-Derived CD133 + Endothelial Progenitor Cells for Cardiac Repair

This study aim to enhance endothelial differentiation of human embryonic stem cells (hESCs) by transduction of an adenovirus (Ad) vector expressing hVEGF(165) gene (Ad-hVEGF(165) ). Purified hESC-derived CD133(+) endothelial progenitors were transplanted into a rat myocardial infarct model to assess their ability to contribute to heart regeneration. Optimal transduction efficiency with high cell viability was achieved by exposing differentiating hESCs to viral particles at a ratio of 1:500 for 4 h on three consecutive days. Reverse transcription-PCR analysis showed positive upregulation of VEGF, Ang-1, Flt-1, Tie-2, CD34, CD31, CD133 and Flk-1 gene expression in Ad-hVEGF(165) -transduced cells. Additionally, flow cytometric analysis of CD133, a cell surface marker, revealed an approximately fivefold increase of CD133 marker expression in Ad-hVEGF(165)-transduced cells compared with the nontransduced control. Within a rat myocardial infarct model, transplanted CD133(+) endothelial progenitor cells survived and participated, both actively and passively, in the regeneration of the infarcted myocardium, as seen by an approximately threefold increase in mature blood vessel density (13.62 +/- 1.56 vs 5.11 +/- 1.23; p < 0.01), as well as significantly reduced infarct size (28% +/- 8.2% vs 76% +/- 5.6%; p < 0.01) in the transplanted group compared with the culture medium-injected control. There was significant improvement in heart function 6 weeks post-transplantation, as confirmed by regional blood-flow analysis (1.72 +/- 0.612 ml/min/g vs 0.8 +/- 0.256 ml/min/g; p < 0.05), as well as echocardiography assessment of left ventricular ejection fraction (60.855% +/- 7.7% vs 38.22 +/- 8.6%; p < 0.05) and fractional shortening (38.63% +/- 9.3% vs 25.2% +/- 7.11%; p < 0.05). hESC-derived CD133(+) endothelial progenitor cells can be utilized to regenerate the infarcted heart.

Angiogenic properties of sustained release platelet-rich plasma: Characterization in-vitro and in the ischemic hind limb of the mouse

While single growth factor has limitation to induce optimal neovascularization, platelet-rich plasma (PRP) is an autologous reserver of various growth factors. However, little is known about the mechanism of PRP-related neovascularization.The objective of this investigation was to characterize the angiogenic and growth factor content of PRP and to determine, in vitro, its effect on endothelial cell proliferation. Additionally, this experiment sought to determine the effectiveness of different compositions of PRP (solution versus sustained release) on perfusion and neovascularization in a murine model of hind limb ischemia. Different growth factors were measured by enzyme-linked immunosorbent assay (ELISA). In vivo study, we used gelatin hydrogel as a sustained release carrier for growth factors in PRP. We induced hind limb ischemia by excising right femoral artery in wild type C57BL6 mice. After surgery, mice were randomly assigned to four experimental groups; control (C), 100 muL of sustained release form of platelet-poor plasma (PPP), 100 muL of solution form of PRP (PRP-sol), 100 muL of sustained release form of PRP (PRP-sr); each formulation was administered via an intramuscular injection to the ischemic hind limb. Endpoint evaluations were blood perfusion by laser Doppler perfusion image, vascular density by anti Von Willebrand factor (vWF), and mature vessel density by anti smooth muscle actin (SMA) antibody. Green fluorescent protein (GFP+) transgenic mice were generated by transplantation of bone marrow derived mononuclear cells to wild type C57BL6 mice, and finally CD34+ cell in the ischemic site of transgenic mice was detected by staining with anti-CD34 antibody. In vitro study showed that PRP containing different growth factors induces endothelial cell proliferation and capillary tube formation. In vivo study demonstrated that sustained release of PRP increased perfusion of ischemic tissue as measured by laser Doppler perfusion imaging (LDPI) (57 +/- 12, 56 +/- 9, 72 +/- 7, 98 +/- 4 for groups C, PPP, PRP-sol, and PRP-sr, respectively; P < .05); capillary density (151 +/- 16, 158 +/- 12, 189 +/- 39, 276 +/- 39 for groups C, PPP, PRP-sol, and PRP-sr, respectively, P < .05) and mature vessel density (28 +/- 2, 31 +/- 3, 52 +/- 10, 85 +/- 13 for groups C, PPP, PRP-sol, and PRP-sr, respectively, P < .05) . Sustained release PRP also increases CD34+ cells in the ischemic site of transgenic mice (6 +/- 3 vs 18 +/- 5/mm(2) for groups control and PRP-sr respectively, P < .05). Sustained release of PRP containing potent angiogenic growth factors restores blood perfusion presumably by stimulating angiogenesis, arteriogenesis, as well as vasculogenesis in the mouse hind limb ischemia. PRP is a natural reserver of various growth factors that can be collected autologously and is costeffective. Thus for clinical use, no special considerations concerning antibody formation and infection risk are needed.Some clinical devices to automatically prepare PRP are available at present. PRP are consistently being used clinically inthe department of orthopedics and plastic surgery (oral, maxillary facial) for a long time. On the basis of researchevidence, some publications have reported positive results in either bone or soft tissue healing. However, some researchconcludes that there is no or little benefit from PRP. This is likely due to faster degradation of growth factors in PRP sincesome authors suggest using sustained release form of PRP to deliver optimal effect of PRP. Gelatin hydrogel is also beingused clinically as a slow, sustained release of carrier for growth factors in our center recently.

The influence of the sequential delivery of angiogenic factors from affinity-binding alginate scaffolds on vascularization

This study describes the features of tissue-engineering scaffold capable of sequentially delivering three angiogenic factors. The scaffold consists of alginate-sulfate/alginate, wherein vascular endothelial growth factor (VEGF) platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β1 (TGF-β1) are bound to alginate-sulfate with an affinity similar to that realized upon their binding to heparin. Factor release rate from the scaffold was correlated with the equilibrium binding constants of the factors to the matrix, thus enabling the sequential delivery of VEGF, PDGF-BB and TGF-β1. In alginate scaffolds lacking alginate-sulfate, release of the adsorbed proteins was instantaneous. After subcutaneous implantation for 1 and 3 months in rats, the blood vessel density and percentage of mature vessels were 3-fold greater in the triple factor-bound scaffolds than in the factor-adsorbed or untreated scaffolds. Moreover, vascularization within the triple factor-bound scaffolds was superior to that found in scaffolds delivering only basic fibroblast growth factor. Application of this novel scaffold may be extended to the combined delivery of additional heparin-binding angiogenic factors or combinations of growth factors active in different tissue regeneration processes.

Blood Vessel Maturation in Human Uveal Melanoma: Spatial Distribution of Neovessels and Mature Vasculature

Purpose: The aims of this study are (1) to evaluate the spatial distribution of neovessels and mature vessels in human uveal melanoma tumors and (2) to determine whether vessel maturation is associated with the major indicators for poor prognosis. Methods: Immunohistochemical analyses were performed on human tissue specimens from enucleated eyes (n = 14) to assess total vessels, neovessels, mature vessels, and cell proliferation. Tumor morphology was analyzed by hematoxylin and eosin and modified periodic acid-Schiff (PAS) staining.The spatial distribution of neovessels and mature vessels was analyzed by immunohistochemistry, and correlated with major indicators of poor prognosis (i.e., aggressive PAS patterns, epithelioid cytology, mitotic figures, extraocular extension, anterior tumor location, ciliary body involvement, large tumor size, cell proliferation, and angiogenic activity). Results: Neovesseldensity was greater than mature vessel density in apical (p = 0.17), central (p = 0.036), and peripheral (p = 0.31) regions of the tumors, while mature vessel density was greater than neovessel density in basal areas of the tumor (p = 0.47). This pattern indicated that vessel maturation begins at the base of the tumor and later extends to the peripheral and apical regions. The difference between mature and neovessel densities for the apical (–0.8 ± 1.9) and central areas (–0.8 ± 1.3) of the tumor was significantly higher than the difference obtained for the basal area (0.3 ± 1.6; p = 0.014 and p = 0.012, respectively), indicating a higher density of mature vessels compared to neovessels at the base. Statistical correlations were found between mature vessel density and tumor size (r = 0.48, p = 0.084), cell proliferation (r = 0.62, p = 0.042), and mitotic figures (r = 0.76, p = 0.001). Conclusions: Significant differences exist in the spatial distribution of mature versus neovessels in human uveal melanoma. Vessel maturation is associated with known clinical and pathologic indicators of poor prognosis (e.g., cell proliferation). Antiangiogenic therapy should be considered for the treatment of ocular malignancies; however, the results of this study indicate that blood vessel maturation heterogeneity may limit the efficacy of vessel targeting agents.