Angiogenesis as therapeutic target has emerged since early works by Judah Folkman, yet his “holy grail” was inhibiting vascular growth to block tumor nutrition. However, in modern biomedicine, “therapeutic angiogenesis” became a large field focusing on stimulation of blood vessel growth for ischemia relief to reduce its detrimental effects in the tissues. In this review, we introduce basic principles of tissue vascularization in response to ischemia exploited in this field. An overview of recent status in therapeutic angiogenesis is given with introduction to emerging technologies, including gene therapy, genetic modification of cells ex vivo and tissue engineering.
- therapeutic angiogenesis
- growth factors
- gene therapy
- cell therapy
- viral vector
Blood vessel growth is a natural process driven by multiple stimuli of which hypoxia is one of the strongest inducing potent response until O2 pressure is normalized by the blood coming through de novo formed vasculature. However, a large group of diseases is caused by hypoxic or ischemic state of tissue. These include peripheral artery disease (PAD) and intermittent claudication (IC), coronary heart disease (CHD), myocardial infarction (MI) and ischemic stroke. Accompanied by endothelial dysfunction and age-related reduction of angiogenic response, they result in disabilities and mortality rate of 25–25% annually. Existing strategies for surgical bypass or endovascular interventions have limited efficacy as far as a cohort of non-option patients expands reaching 25–50% after certain extent of disease progression. Moreover, long-term prognosis after most interventions is negative as grafts undergo restenosis and vascular biocompatible prosthetics are yet to come for wide application. This drew attention of physicians and researchers to the concept of angiogenic therapy to stimulate body’s own resource and form new blood vessels to relieve ischemia. During recent decade the field of biomedicine known as
2. Biological foundations of therapeutic angiogenesis
Postnatal growth of blood vessels is mediated by three mechanisms: vasculogenesis, angiogenesis and arteriogenesis . Vasculogenesis is de novo formation of vasculature from specific progenitor or stem cells; however, it is attributed to prenatal period and after birth its role is unclear  and major extent of blood vessel formation involves two other mechanisms focusing our attention on them. Molecular and cellular basics underlying these processes became the cornerstones of therapeutic angiogenesis and become the source of novel objects for applied researchers and translational medicine.
2.1. Angiogenesis: hypoxia-driven growth of blood vessels
Angiogenesis is the formation of a blood vessel de novo, yet in contrast to vasculogenesis, it relies on migration, proliferation and sprouting of existing endothelial cells (EC) comprising capillaries. The latter are small (8–15 μm) vessels lacking tunica media responsible for majority of tissue blood supply and O2/CO2 exchange . Reduction of tissue O2 induces angiogenesis response in health (intense exercise, tissue growth, etc.) and in disease: in the case of interrupted or declining supply due to atherosclerotic lesions or anemia . Under normal condition, capillaries are stabilized by autocrine and paracrine stimuli (Notch1 axis, angiopoietins, thrombospondin, angiostatin, transforming growth factor (TGF)-β, etc.) that balance influence of pro-angiogenic cytokines within blood vessels’ vicinity (vascular endothelial growth factors (VEGFs), fibroblast growth factors (FGFs), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF)). Hypoxia dislodges this balance toward angiogenic events and this is mediated by O2-sensitive system existing in a variety of cells including EC themselves, smooth muscle cells (SMC), pericytes and fibroblasts. Cells respond to hypoxia via a system of hypoxia-induced factors (HIFs) —a group of heterodimeric transcription regulators controlled by O2-sensitive prolyl hydroxylases. Briefly, stability of HIFs is increased drastically in hypoxic environment resulting in their binding to hypoxia-responsive elements within promoter regions of genes increasing their expression . HIF-dependent genes include a vast array of cytokines stimulating EC proliferation, blood vessel sprouting and, thus, labeled “angiogenic growth factors” [7, 8]. The latter include soluble growth factors associated with EC proliferation and differentiation (acidic FGF (aFGF), basic FGF (bFGF), HGF, VEGFs) [9, 10] and cytokines bound to extracellular matrix (ECM) and released during its cleavage . These changes induce EC proliferation and migration forming a vascular sprout guided by a “tip cell.” This cell follows a gradient of concentration and produces matrix metalloproteinases (MMPs) and urokinase (uPA) to cleave the ECM , releasing growth factors and basically tunneling ECM followed by “stalk cells” that form a new capillary . After lumen formation occurs normalized blood supply switches off hypoxic stimuli, “tip cells” lose their phenotype and proteolytic potential  commencing microenvironment stabilization. Expression of tissue metalloproteinase inhibitors and Dll4-Notch1 axis  induction in EC is followed by reestablishment of a balanced state between pro- and antiangiogenic molecules in the tissue leaving a new capillary-sized blood vessel . However, it should be mentioned that this sequence of events never occurs as a perfectly tuned mechanism. “Stub” branches are formed and must be removed, certain “tip cells” fail to form a sprout and maturation of vascular network includes dissociation of certain anastomoses , which overall describes angiogenesis as a dynamic process modulated by multiple stimuli . Finally, under influence of stabilizing signals from surrounding EC, pericytes and stromal cells, the vascular bed returns to normal steady state.
2.2. Arteriogenesis: shear stress-induced vascular remodeling
Arteriogenesis is triggered by rise of shear stress after an occlusion and induces collateral vessel remodeling forming a bypass 20–100 μm in diameter with developed tunica media. Arteriogenesis may occur gradually (e.g., in increasing stenosis of a large-caliber artery) or can be triggered by a rapidly developed occlusion with both situations are to result in effective blood flow delivery “around an obstacle” to distal portions of the limb or organ . Certain studies show that collateral remodeling can be reversible till certain point of this process in case shear stress drops to normal after thrombolysis or surgical thrombectomy . Effective arteriogenesis may bypass up to 30–40% of basal blood flow in critical stenosis and thrombosis, which is sufficient for tissue survival. However, its efficacy is drastically reduced in disease and with aging . Smoking-related hypercoagulation, hypertension and diabetes also limit arteriogenic response resulting in critical level of ischemia and tissue loss .
After pressure rise in collaterals above the site of thrombosis, shear stress induces EC membrane deformation and flow-sensitive ion channels activate downstream MAP-kinase (ERK1/2, Rho, etc.) phosphorylation and expression of, growth factors, adhesion molecules and chemokines (interleukin-8, macrophage chemoattractant proteins, etc.) . Eventually leukocytes begin to “roll” on EC surface resembling inflammatory changes of vascular function and infiltrate the collateral’s wall . Pivotal role in wall thickening is played by monocytes and their differentiated forms—macrophages and dendritic cells. Their function is not limited to ECM and basal lamina cleavage by MMP and uPA production to destabilize the collateral and make it “flexible” , but they seem to profoundly change the properties of the blood vessel by induction of SMC proliferation and hypertrophy . Under these influences, media thickness may increase 3- to 4-fold and collateral vessel’s volume can enlarge up to 20-fold . Moreover, monocytes produce a wide spectrum of angiogenic and mitogenic cytokines, some of which have antiapoptotic properties required for tissue protection . The role of monocytes and macrophages has been especially emphasized in cardiac arteriogenesis where immunosuppressive steroid hormones , anti-inflammatory therapies and even aspirin  have been shown to negatively impact the outcomes and collateral remodeling. Toxic depletion of monocytes by clodronate reduced arteriogenesis in cryo-injured myocardium and led to decreased ventricular function and higher mortality . As collaterals increase shear stress stimulus is relieved and EC reduce production of chemokines and lose their “adhesive” phenotype. Macrophages limit production of proteolytic enzymes and start ECM reconstruction producing collagens, laminin and elastin and forming adventitial and medial portions of a new arterial vessel.
Typically, we mention “therapeutic angiogenesis” referring gene or cell therapy to relieve ischemia. Nevertheless, one may see that angiogenesis and arteriogenesis share common mediators—namely growth factors and enzymes, ECM components, EC activation, etc. Eventually, for adequate function therapeutic angiogenesis has to rebuild both—medium/large-caliber arteries providing influx of blood and capillary-sized vessels that deliver it to the cells.
3. Therapeutic angiogenesis: methods and approaches
3.1. Protein-based therapeutics
After the discovery of proteins with angiogenic effects, the concept of their therapeutic application was introduced by the 1990s and a vast array of animal studies was published to demonstrate angiogenic efficacy of recombinant protein delivery. Going beyond the VEGF family, experimental works showed induction of angiogenesis by FGFs, HGF, PDGF and placental growth factor (PlGF) in small rodents and rabbits [30, 31]. Injection of these cytokines to ischemic tissue or blood vessels increased perfusion and vascular density. However, promise of this method was questioned as far as achievement of local pharmacological concentration by injection was extremely expensive (especially for human body mass) and half-life of most cytokines was too low to render potent effects . Furthermore, little was known on pharmacokinetics of recombinant proteins delivered intravascularly and their potential involvement in tumor growth and chance of “washout” to systemic blood flow raised safety concerns.
In 2000, the first clinical trials of recombinant human bFGF were initiated in PAD/IC patients after a pilot study showing safety and tolerance of intra-arterial delivery of bFGF solution. Unfortunately, it was halted prior to completion of protocol due to urinalysis data revealing proteinuria in bFGF-treated subjects and no positive changes of endpoints at the moment when the trial was put to a premature end . The final attempt to achieve success in the field was the Therapeutic angiogenesis with recombinant fibroblast growth Factor-2 for intermittent claudication (TRAFFIC) randomized placebo-controlled trial in patients with PAD showing significant improvement in walking time and ankle-brachial index (ABI) in bFGF group. However, safety profile was compromised and yet no cardiac adverse effects or evidence for tumor formation was found in recurrent cases of proteinuria and signs of nephrotoxicity were an issue .
These results were as disappointing as valuable for the field and suggested that gene therapy with its local sustained expression of desired protein is the best alternative possible . Recently, no further attempts to implicate protein delivery for therapeutic angiogenesis were made in clinics and advantages of other methods are exploited to patients’ benefit.
3.2. Gene therapy for angiogenesis
Gene therapy relies on delivery of genetic information by introduction of nucleic acids to target cells/tissues using vector systems. This results in local expression and production of desired protein over a certain period depending on vector used and properties of tissue. First experiments indicating possibility of in vivo gene delivery using simple injection of a recombinant plasmid DNA (pDNA) opened the gate for hundreds of studies published within the last two decades .
As far as the “cornerstone” of gene therapy is the vector system, a brief overview of existing options is required. General concept in the field is that all vectors can be divided into “viral” and “nonviral” subgroups covering nearly any possible way of genetic material delivery. Among nonviral vectors, pDNA is the most widely used due to its long-studied safety profile, ease of production and low immunogenicity allowing repetitive administration [31, 36]. Moreover, plasmids are feasible for combined delivery of several growth factors by mixing them in a formulation or generating a multicistronic vector. However, low transfection efficacy (0.5–2.0% in various tissues) in large mammals including human is an efficacy-limiting issue for pDNA . Viral delivery systems comprise a broad spectrum of recombinant or chimeric viruses of different capacity having a great potential. The latter is due to high transduction efficacy and long expression period accompanied by tissue tropism in certain viruses. However, disadvantages are safety issues: immune reactions and risk of carcinogenesis due to integration to host genome. The most widely spread vectors include adeno- , adeno-associated  and retroviruses, yet in therapeutic angiogenesis, the latter have limited application due to high risk of insertional mutagenesis . Recent progress of molecular engineering allowed development of optimized viral systems exploiting their advantages as well as novel more effective pDNA systems [41, 42].
Period of growth factor-based gene delivery dates back to the seminal study by Dr. J. Isner  who used injection of pDNA encoding VEGF-A 165 (VEGF165) isoform to succeed in treatment of a non-option patient with critical limb ischemia. “First in-human” data were supported by Baumgartner et al. who found increased collateral formation after intramuscular delivery of VEGF165 and EC proliferation in amputation material providing proof of mechanism . Later a number of vectors using VEGF-A and its isoforms were evaluated in experimental and clinical trials making it the most intensively studied object in therapeutic angiogenesis.
Among numerous clinical examples, one may highlight the first “head-to-head” comparison of adenovirus with VEGF165 (Ad-VEGF165) and liposome-packed pDNA-VEGF165 in PAD patients undergoing angioplasty. The trial showed low clinical efficacy of both approaches—Rutherford severity class stayed comparable to control group yet vascular density was increased after treatment . This and other studies using catheter delivery hinted that this method lacks site specificity and intramuscular injection technique was generally adopted. However, initial Groningen double-blind placebo-controlled trial intramuscular injection of pDNA-VEGF165 in PAD patients with diabetes mellitus failed the primary endpoint (amputation rate), yet improvements in ulcer healing, TcO2 and ABI were observed .
Later, Regional angiogenesis with VEGF (RAVE) trial was the first double-blind placebo-controlled trial of VEGF-A 121 isoform (VEGF121). This cytokine is considered to have better solubility than VEGF165 isoform as it lacks a heparin-binding domain . Key feature of this study was an attempt to perform dose optimization of Ad-VEGF121 dividing 105 patients with PAD/IC into three subgroups that received a single session of 20 intramuscular injections of AdVEGF165 delivering low dose (4 × 109 particles), high dose (4 × 1010 particles), or placebo. Final assessment after 12 weeks revealed no significant differences in endpoints between control and treatment subgroups, yet dose-dependent increase of edema adverse effect was observed. Indeed, since first studies delivery of VEGF-A isoforms was haunted by evidence of edema formation due to its influence on endothelial permeability  with certain authors claiming this was a putative reason for low efficacy of therapy .
Trials in MI patients were initiated as early as in 1998 using a pDNA-VEGF165 showing good safety profile and no positive changes . It was followed by Kuopio Angiogenesis Trial  using a comparative design with Ad-VEGF165 or pDNA-VEGF165 delivery by intramyocardial injection during transcutaneous angioplasty. In this trial, no differences between control and treatment groups were found, yet at 6 months after injection of Ad-VEGF165, myocardium perfusion was higher than pDNA-VEGF165, which was attributed to its high transduction efficacy. EuroInject One trial gave similar results showing no significant improvement of myocardial perfusion after injection of pDNA-VEGF165, yet local contractility was higher than control .
Trials using delivery of HGF were initiated and conducted by Dr. Morishita’s group aiming to treat PAD by intramuscular injection of pDNA-HGF. Encouraging results in animal models [31, 53] promoted clinical translation and after safety assessment a Phase II trial was initiated comparing single and repeated dose of pDNA-HGF in favor of multiple injections: only this dosing regimen showed improvement of TcO2 compared to control . Further results in a placebo-controlled I/IIa phase trial showed good safety with no traces of secreted HGF in peripheral blood and repeated injection of 8 mg pDNA-HGF showed significant improvements of secondary endpoints (ulcer size, ABI and pain reduction) . Similar results were obtained in a placebo-controlled trial in PAD patients where by the end of week 12, 70% decrease of ulcer size was observed . Further attempts to increase efficacy included the use of a bicistronic plasmid encoding two forms of HGF named dHGF and cHGF. They were evaluated in animal models showing better perfusion after expression of dHGF + cHGF than each one alone . Clinical trial of this approach in PAD patients showed that multifocal intramuscular injections of 4–16 mg of pDNA-dHGF/cHGF resulted in improvement within 3 months independently of dose: rest and walking pains reduced and a trend toward ulcer healing and increase of TcO2 was observed .
Overall, we may expect HGF-based drugs to become the first widely marketed for PAD—in Japan it has been registered under “Collategene” name and now undergoes stage III clinical trial in PAD cohort. Furthermore, despite HGF has never been tested for MI treatment in clinical settings, preclinical assessments indicate that it may be effective as it has antifibrotic and angiogenic mode of action that can be a good option for this disease or subsequent ventricular failure due to tissue scarring [53, 59].
Fibroblast growth factor has been the first used in protein delivery and gene therapy studies were to follow as soon as it gained attention. Therapeutic angiogenesis leg ischemia study for the management of arteriopathy and non-healing ulcer (TALISMAN-201) have evaluated pDNA-FGF-1 in no-option PAD patients  and showed improvements as decreased amputation rate within 1 year after treatment  and its prospective part showed reduced general mortality in treated subjects . However, phase III placebo-controlled “TAMARIS” (n = 525) trial drew disappointing results and all primary endpoints including amputation events failed to improve after treatment by pDNA-FGF-1 [62, 63]. Similar results obtained in OPTIMIST and EuroOPTIMIST trials indicated safety and lack of efficacy after treatment and lead to wrapping up of this prospective drug testing. Nevertheless, in a follow-up stage, important safety data showing no increased cancer, stroke, or MI in FGF-treated patients was obtained and positively impacted new proceedings in the field .
In MI patients, FGF-4 was delivered using an intracoronary injection of an adenovirus with this gene (Ad-FGF-4) in an Angiogenic gene therapy (AGENT) trial. Result evaluation showed that the only subgroup with reduced size of ischemic myocardium after treatment was female patients when compared to male subgroup. The authors speculated that it may be attributed to higher extent of microcirculatory disorders in females [65, 66] accompanied by fewer critical stenosis typical in men . As far as FGF-4 is known to positively influence endothelial function, this might have been the mechanism for observed changes in the trial. Among other therapeutic factors used for stimulation of angiogenesis, HIF-1α and development endothelial locus-1 (DEL) are both worth a mention as far as they made it to the bedside in recent years using adenovirus or pDNA vectors. However, trials showed minimal improvement in PAD patients and further evaluations were ceased up to date [68, 69].
Overall despite failure to show expected efficacy in clinic, gene therapy is safe and well tolerated by patients showing little evidence although long-term evaluations are yet to be completed. Key obstacle in pDNA-mediated gene therapy relates to transfection efficacy and thus protein production levels after administration . Viral vectors show some promise in solving the problem, yet optimization of dosage regimen, delivery routes and administration protocols also provide a field for further development.
From the point of translational potential, pDNA-based gene therapy has the best safety profile and the best results are definitely yet to come in the following years yet points for improvement are obvious. Efficacy improvement in gene therapy can be achieved by combined approaches basing on the point that angiogenesis is a dynamic process controlled by numerous cytokines, each playing its party in initiation/cessation of different stages. This puts the basis for combined gene therapy to treat ischemia with higher efficacy and it has been supported by experimental findings using VEGF165 combined with another pro-angiogenic growth factor: bFGF , PDGF , angiopoietin-1 , or Stromal cell-derived factor-1α (SDF-1α) . Our previous experience in mouse hind limb ischemia model showed that combination of VEGF165 and uPA  or HGF  induced angiogenic response more effectively than each factor alone or allowed to reduce pDNA dose for combined delivery . A crucial transcription factor in angiogenesis, HIF-1α, was also used for combined gene therapy with VEGF165 showing good results in animal model  as well as bFGF + heme oxygenase-1 (HO-1) . Regarding the latter, it is known that HO-1 is an important regulator of endothelial function with protective function. Its expression is known to induce angiogenesis in ischemic tissues and blockade or knockout reduces EC proliferation and motility and thus capillary growth .
Triple combined gene therapy has not been evaluated for angiogenesis, yet a study was published where controlled release scaffolds containing a mix of VEGF165, HGF and angiopoietin-1 or their double combinations were evaluated for enhancing efficacy of endothelial progenitor cell (EPC) therapy. Triple combinations resulted in significantly higher SMC counts indicating more efficient vessel stabilization due to angiopoietin-1 effects on perivascular cell chemotaxis .
Authors claimed that the use of VEGF165 + another cytokine typically leads to decreased edema and vascular permeability: this has been shown for a well-known stabilizing cytokines—angiopoietin-1  and HGF . Thus, another rationale for combined therapy is decrease of certain “side effects” observed in “monotherapy” by VEGF as a key player in gene therapy. The latter point is not limited to reduction of adverse reactions, but also arises from a large spectrum of pleiotropic effects of cytokines. For example, VEGF may act as a pro-inflammatory cytokine by induction of nuclear factor κ-B, while HGF  or angiopoietin-1 shows antagonistic effects leading to reduction of VEGF-driven cell adhesion and inflammation . Indeed, this has been confirmed in a number of in vitro tests and skin inflammation model indicating that these properties may be utilized for development of next-generation gene therapy drugs for angiogenesis exploiting pleiotropy of cytokines besides their main angiogenic effect. Another approach is delivery of growth factors in two sessions apart in time: for example, pre-treatment by angiopoietin-1 pDNA resulted in better angiogenic response after subsequent pDNA-VEGF165 injection in mouse hind limb ischemia model hinting time of administration as an important factor for efficacy .
3.3. Cell therapy and
ex vivo modified cells
Cell therapy is a promising tool for regenerative medicine and therapeutic angiogenesis using progenitor or stem cells’ ability to self-renew and mediate tissue repair. For potential use of cell therapy for vascular repair, one of the most intriguing findings was the discovery of endothelial progenitor cells (EPC) in circulating blood which hinted involvement of postnatal vasculogenesis in perfusion restoration . However, further works sparkled controversy about EPC phenotype, origin , role in recovery from disease and even existence. Report by Prokopi et al.  claimed that EPC can be false-detected as endothelium-like (CD31/vWF+) monocytes in cultures due to phagocytosis of residual platelets rich with these protein markers.
Clinical trials up to date focus on delivery of bone marrow (BM) cells for induction of angiogenesis. These studies evaluated effects of BM mesenchymal stem cells (BM-MSC) or mononuclear cells (BM-MNC) delivered by intramuscular or intravascular injection in PAD patients. Most studies supported efficacy and indicated improvement in evaluated endpoints: ABI, pain-free walking distance, TcO2, ulcer healing, or amputation-free period. However, some pivotal trials are to be mentioned in detail for better understanding of the field’s status.
First set of crucial data was obtained during “head-to-head” comparison of different cell types to identify the optimal cell source. In a double-blind randomized study, administration of BM-MSC to diabetic PAD patients with foot ulcerations showed efficacy superior to BM-MNC . Subjects that received BM-MSC showed complete ulcer healing 4 weeks earlier than BM-MNC; perfusion assessment, pain-free walking time, ABI and angiography data also spoke in favor of BM-MSC as a more effective cellular angiogenic agent .
However, limitation of BM-based treatment is invasive procedure to obtain material and alternative approach was proposed using peripheral blood mononuclear cells (PB-MNC) mobilized by granulocyte colony-stimulating factor (G-CSF) pre-administration. Feasibility of this approach was obvious and a trial was initiated to confirm its efficacy compared to BM-MNC enrolling a total of 150 patients split in two groups. After 12 weeks of observation, PB-MNC patients showed significantly higher limb temperature, ABI and reduced rest pains than BM-MNC. Yet no difference was found in TcO2, ulcer healing rate and amputation frequency hinting that two methods showed comparable efficacy profile with a trend to PB-MNC application due to feasibility and endpoint improvements . Interestingly, a trial of conventional therapy + G-CSF monotherapy was compared to BM cells and in these groups, improvements in ABI and TcO2 were comparable and significantly better than in conventional drug therapy control. This was an intriguing finding which showed that mobilization of endogenous mononuclear cells (MNC) was sufficient to replace BM grafting and injection .
Another source of cells for therapeutic angiogenesis is adipose-derived mesenchymal stromal cells (AD-MSC). Despite sources of mesenchymal stem cells (MSC) are not limited to adipose tissue, these adult stromal cells can be isolated from samples obtained during lipoaspiration or surgery. Taken together with ease of expansion, well-established phenotype and abundance in healthy individuals, it makes AD-MSC an excellent object for autologous and allogeneic use for angiogenesis stimulation . Published experimental studies show that AD-MSC use their paracrine potential for induction of angiogenesis and support of collateral remodeling . This is referred as “bystander effect” to emphasize that AD-MSC render their effects by paracrine mechanism in contrast to previously existing opinion about their significant ability to differentiate into specific vascular cells and EC in particular .
These cells have not been evaluated in PAD or MI clinical trials yet and considered to be a very attractive option to complement existing strategies. Certain factors limiting potency of AD-MSC exist including donors’ age , comorbidities and effects of
This method has been tested in animal models of ischemia using exogenous delivery of VEGF165 , insulin-like growth factor-1 , HO-1 , or other genes to different types of cells: AD-MSC, EC, BM-MSC, etc. In majority of reports, modification resulted in improvement of response after delivery to ischemic tissue. In our experience, administration of human VEGF165-expressing AD-MSC to ischemic limb of immunodeficient mice resulted in enhanced perfusion and vascular density superior to control cells. Furthermore, muscle necrosis was minimal in this group indicating enhanced blood supply and antiapoptotic effects of VEGF165 as mode of action .
Application of modified stem cells for induction of angiogenesis may be limited in coming years unless safety of modification and full extent of its influence on biological properties of cells is understood.
4. Cell sheets: minimal tissue-engineered constructs
Cell sheets (CS) were first introduced by Dr. Okano’s group and occupied a niche between 3D tissue engineering and 2D cell cultures used to obtain therapeutic cellular materials . Briefly, CS is an attached mono- or multilayered xeno-free construct that consists of viable cells with ECM produced by these cells. Application of this method allowed to circumvent a crucial setback observed in a number of experimental works—poor survival of cells used for therapeutic interventions. One of main reasons for this is procedure of detachment by proteolytic enzymes leading to disruption of ECM (along with deposited cytokines) and loss of intercellular contacts resulting in anoikis and high prevalence of cell death aggravated by passage of cells through a catheter or needle causing mechanical damage. Loss of cells implanted to the tissue by injection in suspended form is estimated as 40–75% within the first 3 days , while CS limits this damage to minimum keeping the cells viable after delivery and enhancing their engraftment . Furthermore, ECM proteins delivered as a part of the construct are known to have a beneficial impact on regeneration and do not have toxic or immunogenic features of chemical or xenogeneic scaffolds. Generation of CS is possible from MSC, fibroblasts, EC, skeletal myoblasts, induced pluripotent stem cells and cardiomyocytes derived from them, BM cells and cardiac progenitor cells —literally, any adherent cell culture after it produces enough ECM to stand mechanical manipulation . CS can be used to cover a significant surface making it a good technique for superficial lesions, cardiomyoplasty and ophthalmologic and microsurgical manipulations. Numerous clinical trials are being run in Japan these years to reveal their full potential in a wide array of disorders .
In relation to angiogenesis, this technique was evaluated in MI models using CS from skeletal myoblasts, AD-MSC, or cardiac progenitor cells showing their ability to generate vascularized additional layer of tissue and facilitate vascular growth in underlying tissue [110, 111]. This resulted in improved ventricular function, limited MI size and fibrosis and favorable outcomes in experimental animals. Comparative study of CS vs. injection of suspended cells showed CS to be superior in terms of most functional and histological endpoints analyzed and using a bioluminescent method, the authors reported higher survival of transplanted rat neonatal cardiomyocytes after CS delivery compared to injection . Recently, clinical application of CS from autologous skeletal myoblasts has begun to treat severe heart failure patients with left ventricular assist devices. Delivery of multilayered constructs resulted in ejection fraction increase sufficient to remove the device and postpone heart transplant as well showing good potential of this approach .
In limb ischemia and diabetes, CS are generally considered to be a tool for ulcer treatment and indeed numerous clinical trials have been initiated within last years. However, our group has been extensively investigating application of CS as an angiogenic therapy in PAD. We have found that subcutaneous delivery of CS from AD-MSC to mice with limb ischemia resulted in robust angiogenic response and CS were superior to dispersed cells in terms of tissue perfusion and vessel density . This piece of evidence provided basis for CS application in PAD indicating that their potential is not limited to cutaneous healing but that paracrine factors are capable to induce angiogenic response in ischemic muscle. Our data were supported almost at the same time in a study by Bak et al. who used mixed CS from SMC and EC for successful treatment of experimental limb ischemia in mice by subcutaneous delivery .
Further improvement of CS potential is possible by application of
Overall, application of CS for therapeutic angiogenesis is a new field and its expansion is expected within next years. These constructs are feasible from a translational point of view as far as they do not contain xenogeneic, artificial, or cadaveric materials circumventing many ethical and safety problems in translation.
5. Concluding remarks
Overall, therapeutic angiogenesis has accumulated a “critical mass” of evidence and approaches that would allow its application in practice within the next 10–15 years expanding the capabilities of treatment. However, possibility to shift from initially used non-option or critical patients may lead to better results in clinical trials, especially in gene therapy, where numerous failures put the whole concept under question several years ago. Development of cell therapy was accompanied by a large framework of regulatory, legal, ethical and industrial work to ensure safety and patients’ benefit. Number of clinical trials is growing every year and fortunately no serious evidence for adverse events or other risks for subjects’ health and well-being was found up-to-date.
Therapeutic angiogenesis has become one of the pioneer methods in translational medicine and its full potential is yet to be unleashed especially in the field of
Study and publication was supported by a grant from Russian Scientific Foundation (RSF) №16-45-03007.
Carmeliet P, Jain RK. Molecular mechanisms and clinical applications of angiogenesis. Nature. 2011;473(7347):298–307.
Beck H, Voswinckel R, Wagner S, Ziegelhoeffer T, Heil M, Helisch A, et al. Participation of bone marrow-derived cells in long-term repair processes after experimental stroke. Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism. 2003;23(6):709–17.
Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nature medicine. 1995;1(1):27–31.
Fong GH. Regulation of angiogenesis by oxygen sensing mechanisms. Journal of molecular medicine (Berlin). 2009;87(6):549–60.
Deveza L, Choi J, Yang F. Therapeutic angiogenesis for treating cardiovascular diseases. Theranostics. 2012;2(8):801–14.
Semenza GL. Oxygen sensing, hypoxia-inducible factors and disease pathophysiology. Annual review of pathology. 2014;9:47–71.
Murakami M, Simons M. Fibroblast growth factor regulation of neovascularization. Current opinion in hematology. 2008;15(3):215–20.
Taimeh Z, Loughran J, Birks EJ, Bolli R. Vascular endothelial growth factor in heart failure. Nature reviews cardiology. 2013;10(9):519–30.
Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nature medicine. 2003;9(6):669–76.
Xin X, Yang S, Ingle G, Zlot C, Rangell L, Kowalski J, et al. Hepatocyte growth factor enhances vascular endothelial growth factor-induced angiogenesis in vitro and in vivo. The American journal of pathology. 2001;158(3):1111–20.
Menshikov M, Torosyan N, Elizarova E, Plakida K, Vorotnikov A, Parfyonova Y, et al. Urokinase induces matrix metalloproteinase-9/gelatinase B expression in THP-1 monocytes via ERK1/2 and cytosolic phospholipase A2 activation and eicosanoid production. Journal of vascular research. 2006;43(5):482–90.
Adams RH, Alitalo K. Molecular regulation of angiogenesis and lymphangiogenesis. Nature reviews molecular cell biology. 2007;8(6):464–78.
Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A, Abramsson A, et al. VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. The journal of cell biology. 2003;161(6):1163–77.
Fischer C, Schneider M, Carmeliet P. Principles and Therapeutic Implications of Angiogenesis, Vasculogenesis and Arteriogenesis. In: Moncada S, Higgs A, editors. The Vascular Endothelium II. Berlin, Heidelberg: Springer Berlin Heidelberg; 2006. p. 157–212.
Hellstrom M, Phng LK, Hofmann JJ, Wallgard E, Coultas L, Lindblom P, et al. Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis. Nature. 2007;445(7129):776–80.
Chung AS, Lee J, Ferrara N. Targeting the tumour vasculature: insights from physiological angiogenesis. Nature reviews cancer. 2010;10(7):505–14.
Jain RK. Molecular regulation of vessel maturation. Nature medicine. 2003;9(6):685–93.
Persson AB, Buschmann IR. Vascular growth in health and disease. Frontiers in molecular neuroscience. 2011;4:14.
Heil M, Eitenmuller I, Schmitz-Rixen T, Schaper W. Arteriogenesis versus angiogenesis: similarities and differences. Journal of cellular and molecular medicine. 2006;10(1):45–55.
Schaper W. Collateral circulation: past and present. Basic research in cardiology. 2009;104(1):5–21.
de Groot D, Pasterkamp G, Hoefer IE. Cardiovascular risk factors and collateral artery formation. European journal of clinical investigation. 2009;39(12):1036–47.
Hillmeister P, Lehmann KE, Bondke A, Witt H, Duelsner A, Gruber C, et al. Induction of cerebral arteriogenesis leads to early-phase expression of protease inhibitors in growing collaterals of the brain. Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism. 2008;28(11):1811–23.
Tkachuk V, Stepanova V, Little PJ, Bobik A. Regulation and role of urokinase plasminogen activator in vascular remodelling. Clinical and experimental pharmacology & physiology. 1996;23(9):759–65.
Parfyonova YV, Plekhanova OS, Tkachuk VA. Plasminogen activators in vascular remodeling and angiogenesis. Biochemistry Biokhimiia. 2002;67(1):119–34.
Scholz D, Ito W, Fleming I, Deindl E, Sauer A, Wiesnet M, et al. Ultrastructure and molecular histology of rabbit hind-limb collateral artery growth (arteriogenesis). Virchows Archiv: an international journal of pathology. 2000;436(3):257–70.
Jaipersad AS, Lip GY, Silverman S, Shantsila E. The role of monocytes in angiogenesis and atherosclerosis. Journal of the American College of Cardiology. 2014;63(1):1–11.
Roberts R, DeMello V, Sobel BE. Deleterious effects of methylprednisolone in patients with myocardial infarction. Circulation. 1976;53(3 Suppl):I204–6.
Duelsner A, Gatzke N, Glaser J, Hillmeister P, Li M, Lee EJ, et al. Acetylsalicylic acid, but not clopidogrel, inhibits therapeutically induced cerebral arteriogenesis in the hypoperfused rat brain. Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism. 2012;32(1):105–14.
van Amerongen MJ, Harmsen MC, van Rooijen N, Petersen AH, van Luyn MJ. Macrophage depletion impairs wound healing and increases left ventricular remodeling after myocardial injury in mice. The American journal of pathology. 2007;170(3):818–29.
Baffour R, Berman J, Garb JL, Rhee SW, Kaufman J, Friedmann P. Enhanced angiogenesis and growth of collaterals by in vivo administration of recombinant basic fibroblast growth factor in a rabbit model of acute lower limb ischemia: dose–response effect of basic fibroblast growth factor. Journal of vascular surgery: official publication, the Society for Vascular Surgery [and] International Society for Cardiovascular Surgery, North American Chapter. 1992;16(2):181–91.
Morishita R, Nakamura S, Hayashi S, Taniyama Y, Moriguchi A, Nagano T, et al. Therapeutic angiogenesis induced by human recombinant hepatocyte growth factor in rabbit hind limb ischemia model as cytokine supplement therapy. Hypertension. 1999;33(6):1379–84.
Grochot-Przeczek A, Dulak J, Jozkowicz A. Therapeutic angiogenesis for revascularization in peripheral artery disease. Gene. 2013;525(2):220–8.
Lazarous DF, Unger EF, Epstein SE, Stine A, Arevalo JL, Chew EY, et al. Basic fibroblast growth factor in patients with intermittent claudication: results of a phase I trial. Journal of the American College of Cardiology. 2000;36(4):1239–44.
Lederman RJ, Mendelsohn FO, Anderson RD, Saucedo JF, Tenaglia AN, Hermiller JB, et al. Therapeutic angiogenesis with recombinant fibroblast growth factor-2 for intermittent claudication (the TRAFFIC study): a randomised trial. Lancet. 2002;359(9323):2053–8.
Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, et al. Direct gene transfer into mouse muscle in vivo. Science. 1990;247(4949 Pt 1):1465–8.
Morishita R, Aoki M, Hashiya N, Makino H, Yamasaki K, Azuma J, et al. Safety evaluation of clinical gene therapy using hepatocyte growth factor to treat peripheral arterial disease. Hypertension. 2004;44(2):203–9.
van Gaal EV, Hennink WE, Crommelin DJ, Mastrobattista E. Plasmid engineering for controlled and sustained gene expression for nonviral gene therapy. Pharmaceutical research. 2006;23(6):1053–74.
Khare R, Chen CY, Weaver EA, Barry MA. Advances and future challenges in adenoviral vector pharmacology and targeting. Current gene therapy. 2011;11(4):241–58.
Dismuke DJ, Tenenbaum L, Samulski RJ. Biosafety of recombinant adeno-associated virus vectors. Current gene therapy. 2013;13(6):434–52.
Verma IM, Weitzman MD. Gene therapy: twenty-first century medicine. Annual review of biochemistry. 2005;74:711–38.
MacColl GS, Novo FJ, Marshall NJ, Waters M, Goldspink G, Bouloux PM. Optimisation of growth hormone production by muscle cells using plasmid DNA. Journal of endocrinology. 2000;165(2):329–36.
Makarevich PI, Rubina KA, Diykanov DT, Tkachuk VA, Parfyonova YV. Therapeutic angiogenesis using growth factors: current state and prospects for development. Kardiologiia. 2015;55(9):59–71.
Takeshita S, Zheng LP, Brogi E, Kearney M, Pu LQ, Bunting S, et al. Therapeutic angiogenesis. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model. The journal of clinical investigation. 1994;93(2):662–70.
Baumgartner I, Pieczek A, Manor O, Blair R, Kearney M, Walsh K, et al. Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia. Circulation. 1998;97(12):1114–23.
Makinen K, Manninen H, Hedman M, Matsi P, Mussalo H, Alhava E, et al. Increased vascularity detected by digital subtraction angiography after VEGF gene transfer to human lower limb artery: a randomized, placebo-controlled, double-blinded phase II study. Molecular therapy: the journal of the American Society of Gene Therapy. 2002;6(1):127–33.
Kusumanto YH, van Weel V, Mulder NH, Smit AJ, van den Dungen JJ, Hooymans JM, et al. Treatment with intramuscular vascular endothelial growth factor gene compared with placebo for patients with diabetes mellitus and critical limb ischemia: a double-blind randomized trial. Human gene therapy. 2006;17(6):683–91.
Rajagopalan S, Mohler ER, 3rd, Lederman RJ, Mendelsohn FO, Saucedo JF, Goldman CK, et al. Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease: a phase II randomized, double-blind, controlled study of adenoviral delivery of vascular endothelial growth factor 121 in patients with disabling intermittent claudication. Circulation. 2003;108(16):1933–8.
Vajanto I, Rissanen TT, Rutanen J, Hiltunen MO, Tuomisto TT, Arve K, et al. Evaluation of angiogenesis and side effects in ischemic rabbit hindlimbs after intramuscular injection of adenoviral vectors encoding VEGF and LacZ. The journal of gene medicine. 2002;4(4):371–80.
Shimamura M, Nakagami H, Koriyama H, Morishita R. Gene therapy and cell-based therapies for therapeutic angiogenesis in peripheral artery disease. BioMed research international. 2013;2013:186215.
Losordo DW, Vale PR, Symes JF, Dunnington CH, Esakof DD, Maysky M, et al. Gene therapy for myocardial angiogenesis: initial clinical results with direct myocardial injection of phVEGF165 as sole therapy for myocardial ischemia. Circulation. 1998;98(25):2800–4.
Hedman M, Hartikainen J, Syvanne M, Stjernvall J, Hedman A, Kivela A, et al. Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemia: phase II results of the Kuopio Angiogenesis Trial (KAT). Circulation. 2003;107(21):2677–83.
Gyongyosi M, Khorsand A, Zamini S, Sperker W, Strehblow C, Kastrup J, et al. NOGA-guided analysis of regional myocardial perfusion abnormalities treated with intramyocardial injections of plasmid encoding vascular endothelial growth factor A-165 in patients with chronic myocardial ischemia: subanalysis of the EUROINJECT-ONE multicenter double-blind randomized study. Circulation. 2005;112(9 Suppl):I157–65.
Aoki M, Morishita R, Taniyama Y, Kida I, Moriguchi A, Matsumoto K, et al. Angiogenesis induced by hepatocyte growth factor in non-infarcted myocardium and infarcted myocardium: up-regulation of essential transcription factor for angiogenesis, ets. Gene therapy. 2000;7(5):417–27.
Morishita R, Aoki M, Hashiya N, Yamasaki K, Kurinami H, Shimizu S, et al. Therapeutic angiogenesis using hepatocyte growth factor (HGF). Current gene therapy. 2004;4(2):199–206.
Morishita R, Makino H, Aoki M, Hashiya N, Yamasaki K, Azuma J, et al. Phase I/IIa clinical trial of therapeutic angiogenesis using hepatocyte growth factor gene transfer to treat critical limb ischemia. Arteriosclerosis, thrombosis and vascular biology. 2011;31(3):713–20.
Shigematsu H, Yasuda K, Iwai T, Sasajima T, Ishimaru S, Ohashi Y, et al. Randomized, double-blind, placebo-controlled clinical trial of hepatocyte growth factor plasmid for critical limb ischemia. Gene therapy. 2010;17(9):1152–61.
Pyun WB, Hahn W, Kim DS, Yoo WS, Lee SD, Won JH, et al. Naked DNA expressing two isoforms of hepatocyte growth factor induces collateral artery augmentation in a rabbit model of limb ischemia. Gene therapy. 2010;17(12):1442–52.
Gu Y, Zhang J, Guo L, Cui S, Li X, Ding D, et al. A phase I clinical study of naked DNA expressing two isoforms of hepatocyte growth factor to treat patients with critical limb ischemia. The journal of gene medicine. 2011;13(11):602–10.
Taniyama Y, Morishita R, Nakagami H, Moriguchi A, Sakonjo H, Shokei K, et al. Potential contribution of a novel antifibrotic factor, hepatocyte growth factor, to prevention of myocardial fibrosis by angiotensin II blockade in cardiomyopathic hamsters. Circulation. 2000;102(2):246–52.
Nikol S, Baumgartner I, Van Belle E, Diehm C, Visona A, Capogrossi MC, et al. Therapeutic angiogenesis with intramuscular NV1FGF improves amputation-free survival in patients with critical limb ischemia. Molecular therapy: the journal of the American Society of Gene Therapy. 2008;16(5):972–8.
Comerota AJ, Throm RC, Miller KA, Henry T, Chronos N, Laird J, et al. Naked plasmid DNA encoding fibroblast growth factor type 1 for the treatment of end-stage unreconstructible lower extremity ischemia: preliminary results of a phase I trial. Journal of vascular surgery: official publication, the Society for Vascular Surgery [and] International Society for Cardiovascular Surgery, North American Chapter. 2002;35(5):930–6.
Fowkes FG, Price JF. Gene therapy for critical limb ischaemia: the TAMARIS trial. Lancet. 2011;377(9781):1894–6.
Belch J, Hiatt WR, Baumgartner I, Driver IV, Nikol S, Norgren L, et al. Effect of fibroblast growth factor NV1FGF on amputation and death: a randomised placebo-controlled trial of gene therapy in critical limb ischaemia. Lancet. 2011;377(9781):1929–37.
Niebuhr A, Henry T, Goldman J, Baumgartner I, van Belle E, Gerss J, et al. Long-term safety of intramuscular gene transfer of non-viral FGF1 for peripheral artery disease. Gene therapy. 2012;19(3):264–70.
Reis SE, Holubkov R, Conrad Smith AJ, Kelsey SF, Sharaf BL, Reichek N, et al. Coronary microvascular dysfunction is highly prevalent in women with chest pain in the absence of coronary artery disease: results from the NHLBI WISE study. American heart journal. 2001;141(5):735–41.
Handberg E, Johnson BD, Arant CB, Wessel TR, Kerensky RA, von Mering G, et al. Impaired coronary vascular reactivity and functional capacity in women: results from the NHLBI Women's Ischemia Syndrome Evaluation (WISE) Study. Journal of the American College of Cardiology. 2006;47(3 Suppl):S44–9.
Hochman JS, Tamis JE, Thompson TD, Weaver WD, White HD, Van de Werf F, et al. Sex, clinical presentation and outcome in patients with acute coronary syndromes. Global Use of Strategies to Open Occluded Coronary Arteries in Acute Coronary Syndromes IIb Investigators. The New England journal of medicine. 1999;341(4):226–32.
Yonemitsu Y, Matsumoto T, Itoh H, Okazaki J, Uchiyama M, Yoshida K, et al. DVC1-0101 to treat peripheral arterial disease: a Phase I/IIa open-label dose-escalation clinical trial. Molecular therapy: the journal of the American Society of Gene Therapy. 2013;21(3):707–14.
Creager MA, Olin JW, Belch JJ, Moneta GL, Henry TD, Rajagopalan S, et al. Effect of hypoxia-inducible factor-1 alpha gene therapy on walking performance in patients with intermittent claudication. Circulation. 2011;124(16):1765–73.
Yla-Herttuala S, Markkanen JE, Rissanen TT. Gene therapy for ischemic cardiovascular diseases: some lessons learned from the first clinical trials. Trends in cardiovascular medicine. 2004;14(8):295–300.
Spanholtz TA, Theodorou P, Holzbach T, Wutzler S, Giunta RE, Machens HG. Vascular endothelial growth factor (VEGF165) plus basic fibroblast growth factor (bFGF) producing cells induce a mature and stable vascular network--a future therapy for ischemically challenged tissue. The journal of surgical research. 2011;171(1):329–38.
Kupatt C, Hinkel R, Pfosser A, El-Aouni C, Wuchrer A, Fritz A, et al. Cotransfection of vascular endothelial growth factor-A and platelet-derived growth factor-B via recombinant adeno-associated virus resolves chronic ischemic malperfusion role of vessel maturation. Journal of the American College of Cardiology. 2010;56(5):414–22.
Arsic N, Zentilin L, Zacchigna S, Santoro D, Stanta G, Salvi A, et al. Induction of functional neovascularization by combined VEGF and angiopoietin-1 gene transfer using AAV vectors. Molecular therapy: the journal of the American Society of Gene Therapy. 2003;7(4):450–9.
Yu JX, Huang XF, Lv WM, Ye CS, Peng XZ, Zhang H, et al. Combination of stromal-derived factor-1 alpha and vascular endothelial growth factor gene-modified endothelial progenitor cells is more effective for ischemic neovascularization. Journal of vascular surgery: official publication, the Society for Vascular Surgery [and] International Society for Cardiovascular Surgery, North American Chapter. 2009;50(3):608–16.
Traktuev DO, Tsokolaeva ZI, Shevelev AA, Talitskiy KA, Stepanova VV, Johnstone BH, et al. Urokinase gene transfer augments angiogenesis in ischemic skeletal and myocardial muscle. Molecular therapy: the journal of the American Society of Gene Therapy. 2007;15(11):1939–46.
Makarevich P, Tsokolaeva Z, Shevelev A, Rybalkin I, Shevchenko E, Beloglazova I, et al. Combined transfer of human VEGF165 and HGF genes renders potent angiogenic effect in ischemic skeletal muscle. PLoS one. 2012;7(6):e38776.
Lee S, Kim K, Kim HA, Kim SW, Lee M. Augmentation of erythropoietin enhancer-mediated hypoxia-inducible gene expression by co-transfection of a plasmid encoding hypoxia-inducible factor 1 alpha for ischemic tissue targeting gene therapy. Journal of drug targeting. 2008;16(1):43–50.
Bhang SH, Kim JH, Yang HS, La WG, Lee TJ, Sun AY, et al. Combined delivery of heme oxygenase-1 gene and fibroblast growth factor-2 protein for therapeutic angiogenesis. Biomaterials. 2009;30(31):6247–56.
Dulak J, Jozkowicz A, Foresti R, Kasza A, Frick M, Huk I, et al. Heme oxygenase activity modulates vascular endothelial growth factor synthesis in vascular smooth muscle cells. Antioxidants & redox signaling. 2002;4(2):229–40.
Saif J, Schwarz TM, Chau DY, Henstock J, Sami P, Leicht SF, et al. Combination of injectable multiple growth factor-releasing scaffolds and cell therapy as an advanced modality to enhance tissue neovascularization. Arteriosclerosis, thrombosis and vascular biology. 2010;30(10):1897–904.
Yang Y, Chen QH, Liu AR, Xu XP, Han JB, Qiu HB. Synergism of MSC-secreted HGF and VEGF in stabilising endothelial barrier function upon lipopolysaccharide stimulation via the Rac1 pathway. Stem cell research & therapy. 2015;6:250.
Min JK, Lee YM, Kim JH, Kim YM, Kim SW, Lee SY, et al. Hepatocyte growth factor suppresses vascular endothelial growth factor-induced expression of endothelial ICAM-1 and VCAM-1 by inhibiting the nuclear factor-kappaB pathway. Circulation research. 2005;96(3):300–7.
Kim I, Moon SO, Park SK, Chae SW, Koh GY. Angiopoietin-1 reduces VEGF-stimulated leukocyte adhesion to endothelial cells by reducing ICAM-1, VCAM-1 and E-selectin expression. Circulation research. 2001;89(6):477–9.
Yamauchi A, Ito Y, Morikawa M, Kobune M, Huang J, Sasaki K, et al. Pre-administration of angiopoietin-1 followed by VEGF induces functional and mature vascular formation in a rabbit ischemic model. The journal of gene medicine. 2003;5(11):994–1004.
Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997;275(5302):964–7.
Rohde E, Malischnik C, Thaler D, Maierhofer T, Linkesch W, Lanzer G, et al. Blood monocytes mimic endothelial progenitor cells. Stem cells. 2006;24(2):357–67.
Prokopi M, Pula G, Mayr U, Devue C, Gallagher J, Xiao Q, et al. Proteomic analysis reveals presence of platelet microparticles in endothelial progenitor cell cultures. Blood. 2009;114(3):723–32.
Lu D, Chen B, Liang Z, Deng W, Jiang Y, Li S, et al. Comparison of bone marrow mesenchymal stem cells with bone marrow-derived mononuclear cells for treatment of diabetic critical limb ischemia and foot ulcer: a double-blind, randomized, controlled trial. Diabetes research and clinical practice. 2011;92(1):26–36.
Huang PP, Yang XF, Li SZ, Wen JC, Zhang Y, Han ZC. Randomised comparison of G-CSF-mobilized peripheral blood mononuclear cells versus bone marrow-mononuclear cells for the treatment of patients with lower limb arteriosclerosis obliterans. Thrombosis and haemostasis. 2007;98(6):1335–42.
Arai M, Misao Y, Nagai H, Kawasaki M, Nagashima K, Suzuki K, et al. Granulocyte colony-stimulating factor: a noninvasive regeneration therapy for treating atherosclerotic peripheral artery disease. Circulation journal: official journal of the Japanese Circulation Society. 2006;70(9):1093–8.
Bourin P, Bunnell BA, Casteilla L, Dominici M, Katz AJ, March KL, et al. Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT). Cytotherapy. 2013;15(6):641–8.
Rubina K, Kalinina N, Efimenko A, Lopatina T, Melikhova V, Tsokolaeva Z, et al. Adipose stromal cells stimulate angiogenesis via promoting progenitor cell differentiation, secretion of angiogenic factors and enhancing vessel maturation. Tissue engineering Part A. 2009;15(8):2039–50.
Yang D, Wang W, Li L, Peng Y, Chen P, Huang H, et al. The relative contribution of paracrine effect versus direct differentiation on adipose-derived stem cell transplantation mediated cardiac repair. PLoS one. 2013;8(3):e59020.
Efimenko A, Starostina E, Kalinina N, Stolzing A. Angiogenic properties of aged adipose derived mesenchymal stem cells after hypoxic conditioning. Journal of translational medicine. 2011;9:10.
Efimenko A, Dzhoyashvili N, Kalinina N, Kochegura T, Akchurin R, Tkachuk V, et al. Adipose-derived mesenchymal stromal cells from aged patients with coronary artery disease keep mesenchymal stromal cell properties but exhibit characteristics of aging and have impaired angiogenic potential. Stem cells translational medicine. 2014;3(1):32–41.
Makarevich PI, Dergilev KV, Tsokolaeva ZI, Efimenko AY, Gluhanuk EV, Gallinger JO, et al. Delivery of genetically engineered adipose-derived cell sheets for treatment of ischemic disorders-development of application in animal models. Molecular therapy; Nature Publishing Group: New York, NY, USA; 2015. p. S262-S.
Merdan T, Kopecek J, Kissel T. Prospects for cationic polymers in gene and oligonucleotide therapy against cancer. Advanced drug delivery reviews. 2002;54(5):715–58.
Shevchenko EK, Makarevich PI, Tsokolaeva ZI, Boldyreva MA, Sysoeva VY, Tkachuk VA, et al. Transplantation of modified human adipose derived stromal cells expressing VEGF165 results in more efficient angiogenic response in ischemic skeletal muscle. Journal of translational medicine. 2013;11:138.
Sen S, Merchan J, Dean J, Ii M, Gavin M, Silver M, et al. Autologous transplantation of endothelial progenitor cells genetically modified by adeno-associated viral vector delivering insulin-like growth factor-1 gene after myocardial infarction. Human gene therapy. 2010;21(10):1327–34.
Suzuki M, Iso-o N, Takeshita S, Tsukamoto K, Mori I, Sato T, et al. Facilitated angiogenesis induced by heme oxygenase-1 gene transfer in a rat model of hindlimb ischemia. Biochemical and biophysical research communications. 2003;302(1):138–43.
Holzinger A, Barden M, Abken H. The growing world of CAR T cell trials: a systematic review. Cancer Immunol Immunother. 2016;65(12):1433–50.
Phase I study of IM injection of VEGF-producing MSC for the treatment of critical limb ischemia [Available from: https://www.cirm.ca.gov/our-progress/awards/phase-i-study-im-injection-vegf-producing-msc-treatment-critical-limb-ischemia-0.
Papapetrou EP, Lee G, Malani N, Setty M, Riviere I, Tirunagari LM, et al. Genomic safe harbors permit high beta-globin transgene expression in thalassemia induced pluripotent stem cells. Nature biotechnology. 2011;29(1):73–8.
Kumashiro Y, Fukumori K, Takahashi H, Nakayama M, Akiyama Y, Yamato M, et al. Modulation of cell adhesion and detachment on thermo-responsive polymeric surfaces through the observation of surface dynamics. Colloids and surfaces B: biointerfaces. 2013;106:198–207.
Aguado BA, Mulyasasmita W, Su J, Lampe KJ, Heilshorn SC. Improving viability of stem cells during syringe needle flow through the design of hydrogel cell carriers. Tissue engineering Part A. 2012;18(7–8):806–15.
Lin CY, Lin KJ, Li KC, Sung LY, Hsueh S, Lu CH, et al. Immune responses during healing of massive segmental femoral bone defects mediated by hybrid baculovirus-engineered ASCs. Biomaterials. 2012;33(30):7422–34.
Dergilev K, Tsokolaeva Z, Rubina K, Sysoeva V, Makarevich P, Boldyreva M, et al. Isolation and characterization of cardiac progenitor cells from myocardial right atrial appendage tissue. Cell and tissue biology. 2016;10(5):349–56.
Elloumi-Hannachi I, Yamato M, Okano T. Cell sheet engineering: a unique nanotechnology for scaffold-free tissue reconstruction with clinical applications in regenerative medicine. Journal of internal medicine. 2010;267(1):54–70.
Matsuura K, Utoh R, Nagase K, Okano T. Cell sheet approach for tissue engineering and regenerative medicine. Journal of controlled release: official journal of the Controlled Release Society. 2014;190:228–39.
Yang J, Yamato M, Shimizu T, Sekine H, Ohashi K, Kanzaki M, et al. Reconstruction of functional tissues with cell sheet engineering. Biomaterials. 2007;28(34):5033–43.
Yang J, Yamato M, Kohno C, Nishimoto A, Sekine H, Fukai F, et al. Cell sheet engineering: recreating tissues without biodegradable scaffolds. Biomaterials. 2005;26(33):6415–22.
Sekine H, Shimizu T, Dobashi I, Matsuura K, Hagiwara N, Takahashi M, et al. Cardiac cell sheet transplantation improves damaged heart function via superior cell survival in comparison with dissociated cell injection. Tissue engineering Part A. 2011;17(23–24):2973–80.
Mitamura Y. Current status of left ventricular assist devices and cell sheet engineering for treatment of severe heart disease in Japan. Artificial organs. 2015;39(7):543–9.
Makarevich P, Boldyreva M, Dergilev K, Gluhanyuk E, Gallinger J, Efimenko A, et al. Transplantation of cell sheets from adipose-derived mesenchymal stromal cells effectively induces angiogenesis in ischemic skeletal muscle. Genes and cells. 2015;10(3):68–77.
Bak S, Ahmad T, Lee YB, Lee JY, Kim EM, Shin H. Delivery of a cell patch of cocultured endothelial cells and smooth muscle cells using thermoresponsive hydrogels for enhanced angiogenesis. Tissue engineering Part A. 2016;22(1–2):182–93.
Yeh TS, Fang YH, Lu CH, Chiu SC, Yeh CL, Yen TC, et al. Baculovirus-transduced, VEGF-expressing adipose-derived stem cell sheet for the treatment of myocardium infarction. Biomaterials. 2014;35(1):174–84.