Peripheral Arterial Disease

Abstract

Peripheral Arterial Disease (PAD) is currently known to be a critical cause of mortality and morbidity both in the developing and the developed economy’s countries. Despite the fact that the risk for forces for PAD are thoroughly defined and established PAD patients who in the period acquires Critical Lower Limb Ischemia (CLI) are in often cases asymptotic before the growth of CLI thus the prime forces that determine these patients outcomes are currently not apparent. Despite the advancement of treatment options for these patients, the mortality rate is particularly 25 percent where about 30 percent are required to acquire amputation. The study established that all the approaches utilized with the objective of raising the unique pro-angiogenic expression force lead to clinical failure. This paper will, therefore, focus on analyzing suitable therapies that can be utilized in inducing neovascularization in reducing procedural failure and high mortality rate.

Background and Introduction

Peripheral Arterial Disease is highly predominant, increasingly recognized and debilitating condition, estimated to affect more than 25 million individuals in western countries (Criqui, 1985 264). CLI is categorized to be the final PAD stage. It is can best be described as a medical disorder where patients with Obstructive Arterial Disease, particularly of the lower Limbs, normally acquire Chronic Ischemia resting pain, foot gangrene or ulcers (Norgren, 2007 433). The general number of patients diagnosed with PAD CLI is approximated to be amid ×and 3 percent while the annual number in America is projected to be 160,000 persons (Becker, 2011 1584). CLI patients normally accommodate numerous comorbidities alongside DM while smoking is the most essential, adding to the general high mortality level and the associate apparent economic liability (Criqui, 2001 1583). Despite the adoption of the most suitable medical treatment, which incorporates restricted risk factors control for methodical atherosclerosis, the most effective treatment standard and the only Limb treatment option in reference to CLI remains to be Revascularization.

Lower Limb Revascularization can be carried out either by conventional surgery, percutaneous endovascular approach or by a hybrid procedure which is both surgical and endovascular. Taking into account PAD´s complexity, one patient one anatomical disease pattern, the accessibility guidelines and above all factors, such as, the availability of an autologous conduit for bypass surgery, a patient-tailored approach is probably the best way for success in treating these patients. Choosing the proper therapeutic option may lead to limb salvage and survival rates above 75% in the first year post-diagnosis. However, despite the most recent years which have been characterized by multiple technological discoveries and the intuition of special and intensive care treatment units for these category of patients the mortality rate for a single year is projected to be nearly 25 percent with more 30 percent of the ailing populace necessitating  amputation (Norgren, 2007 433). The survival level for a 5 year period for the CLI patients is stated to be lower than 30 percent (Adam, 2005 812), (Hirsch, 2006 453). In addition, amongst the patients that undergo amputation around 30 percent of the populace necessitates repeated surgeries in the unchanged or on the contralateral leg within a span of 2 years (Norgren, 2007 433).

In approximately 20 to 30 percent of patients diagnosed with CLI, the distribution, comorbidities as well as arterial occlusions diffuseness leads them to be accounted as candidates that are not qualified for either endovascular or surgical revascularization procedures thus amputation is usually the only viable and available choice. Moreover, individuals with renal chronic failure as well as DM and develops CLI holds a poor Salvage Prognosis for the Limbs and it is even reasoned by a number of researchers that prime amputation is the only suitable therapeutic choice for these patient group (Sigala, 2006 1589) and (Venermo, 2011 1592). In other words, poor medical CLI prognosis, the rising CLI patient’s number, prior revascularization failure procedures and the considerable non-option CLI patient’s number have generated a growing necessity for fresh therapies to neovascularization induction. Therapeutic angiogenesis, with the most emphasis being placed on gene and cell therapy, focusing on the use of exogenous molecular and cellular agents to promote revascularization or regeneration of diseased microvasculature.

Literature Review

Even though preclinical studies and early clinical trials regarding therapeutic angiogenesis have presented promising results, single dose administration of proteins did not show lasting effects, probably because of the short half-lives of the proteins administered. Gene therapy with DNA plasmids encoding pro-angiogenic factors has been developed in order to increase the duration of transgenic expression as compared to direct administration of proteins. Despite the plasmids being adequately endured by the bodily immune system they normally result in reduced genes transfer levels with reduced transgenic expression duration. This, therefore, demonstrates that all the approaches utilized with the objective of raising the unique pro-angiogenic expression force lead to clinical failure.

    In the recent cellular therapy has gained preponderance. However, it cannot forget that DAP is associated with endothelial dysfunction and a significant reduction in the number of circulating EPCs and their function (Hill, 2003 1608). Peripheral arterial disease EPCs may not respond to pro-angiogenic factors, particularly in aged patients with DM or hypercholesterolemia (Isner, 1999 950) and (Rivard, 1999 1607). Also, concerning cell-based therapeutic strategies challenges still include questions of cell source, phenotype, preparation protocols, dosing, route and frequency of administration. In short, results shorter than expected from the more recent clinical trials may be explained as a consequence of limitations related to: (i) animal models used in basic research; (ii) patient selection and issues related to different steps in the preparation and administration of genes or stem cells; and (iii) finally, we must take into account the low physiological reserve of the critical patient suffering from a life-threatening disease (Wahlberg, 2003 1271).

    Radiation ionization, in doses that range from 2 up to 8 GY has been established to be promoting angiogenesis most often through raising pro-angiogenic expression forces like IL5, IL4, IL3, TGFβ, IL1Ra, VEGF, FGF, and IL10. This is driven by the cells tumor and micro- surrounding activation with the inclusion of vasculature (Moeller, 2004 1521), (Park, 2006 1609), (Sonveaux, 2003 1106), (Hlatky, 1994 1637), (McBride, 2004 974) and (Yoshimura, 2013 1617). Provided with the extensive utilization of radiotherapy as Malignant tumors treatment option, research has mainly been focused on gaining a more improved understanding on the grounds via which tumor vasculature is activated by IR therapeutic doses in addition to the irradiated micro surrounding  metastasis and invasion(McBride, 2004 974) and (Ciric, 2010 1643). Our research lab has demonstrated for the first time that LDIR, defined as doses lower than 0.8 Gy, induce a pro-angiogenic phenotype in ECs in vitro modulating endothelial dysfunction, promoting survival, migration and preventing EC apoptosis (Sofia Vala, 2010 938). Likewise, we showed that LDIR promotes neovascularization in vivo by inducing angiogenic sprouting in the transgenic fluorescent zebrafish Tg. Such operations mainly focused on an innovative strategy, for the vasculature that covers the tumor and acquires LDIR relativity during treatment procedures.

            The established findings were justified by the acquired data from a microarray research where several proteins encoding transcripts necessitated for procedural angiogenesis were prompted after LDIR delivery. The revelation by a global gene expression analysis demonstrated that 2374 genes were modulated by LDIR and from those, 1344, many of which with a role in angiogenesis, were up-regulated in LDIR versus control Human Lung Microvascular Endothelial Cells (HMVEC-L) at a cutoff corresponding to a P value<0.03. The biological relevance of these data was transduced in the most prominent signaling pathways related to a molecular LDIR response associated, primarily to three distinct but significant groups. 1) Development force and signaling Cytokine which regulates the proliferation, survival, distinctiveness, and migration. This incorporates TGFβ2 signaling and Epidermal Growth Factor (EGF) (Distler, 2003 667) and (Kuwano, 2001 1066). 2) Cytoskeleton-associated components involved in the arrangement of cytoskeleton, migration, and adhesion (Adams, 2010 1068), (Chien, 2005 1070), (Hashimoto-Torii, 2008 1071), (Lamalice, 2007 1073) and (Munoz-Chapuli, 2004 1074). 3) Stress-projected molecules that are mainly linked with IR projected damages. These damages play a crucial responsibility in the progression of cells and checkpoint DNA repair (Pawlik, 2004 1077).

            For those genes that their expression is altered significantly byLDIR and are a representation of the most suitable proangiogenic candidate's responses were chosen. These are VEGFR1 and VEGFR2, ANG2, TGFβ, PDGF, and FGF-2. Hepatocyte development factor as well as the respective receptor, c-MET were also validated as HGF use which has been projected in therapeutic angiogenesis setting as well as HGF medical trials gene therapies. The expressions were thus validated by real-time quantitative PCR as well as Western Blot with the utilization of non-irradiates and irradiated HMVEC-L. As soon as the 4th-hour post0.3 GY exposure majority’s expression of the chosen pro-angiogenic molecules were then risen then returned to the 12h baseline after LDIR. The short-run LDIR impact on ECs is generally independent of all fractionation doses. This is because cells that are exposed to 0.3 GY given for two, three or even four days consecutively that demonstrated similar expression gene pattern as well as magnitude.

Further, it was established that LDICR normally activates the Endothelium through phosphorylation of VEGFR2 which is an essential angiogenic process player (Sofia Vala, 2010 938). Based on the results it can thus be described can VEGFR2 activation results in speedy activation of differing cellular proteins and correspondingly to mRNA de novo and the expression of protein mediators that are incorporated in angiogenic responses (Zachary, 2003 1383) and (Zachary, 2001 1623). Accordingly, the signaling pathways like Threonine/Serine and Signal extracellular controlled Mitogen Kinase protein are thus activated while the modulation of gene expression occurs.

Results/Discussion

            Given these findings, we aimed at testing an innovative non-invasive strategy, using LDIR to induce therapeutic angiogenesis in vivo in a murine HLIM.

            Preclinical studies aimed at evaluating the efficacy of potential new pharmacological agents in the field of therapeutic angiogenesis include several categories of both In-vivo and In-vitro evaluations. EC tubule creation evaluation is mainly significant for the new therapeutic components screening given that it is efficient on cost, not demanding technically, suitable to the extensive scrutiny scale and permits the testing of cells derived from humans (Staton, 2009 1650). However, an in vivo model translates in a more precise way the high complexity of the human body; the vasculature of the skeletal muscle is a three-dimensional structure that includes capillaries and larger vessels consisting of pericytes, ECs, monocytes as well as leveled muscle cells that associated with the matrix extracellular. Consequently, neovascularisation signifies the harmonious association of these components with the micro surrounding (Staton, 2009 1650) and (Hudlicka, 1992 1652). As per the current, animal based limbs approaches ischemia are accounted to being the most suitable CLI conditions mimic, in the local hemodynamics like shear stress and blood flow, determine limb-generated response to ischemia as well as to the therapeutic agents under study.

            HLIM normally incorporates arterial supply acute interruption while it remains to be the most utilized pre-medical in-vivo strategy for angiogenic as well as arteriogenic evaluation of the possible agents and respective cells (Aranguren, 2009 1653) and (Hudlicka, 1994 1654). Rodent tends to be the most often utilized but regardless porcine, primate, rabbit and canine models have additionally been discussed. The utilization of small sized animals like rodents specifically the mouse holds advantages based on the increased transgenic strains availability and experimentation reduced cost. Despite HLIM being accounted to be associated with more medical relevance in relation to PAD and particularly CLI, it does not completely generate the human condition that has complexity. There are differing HLMs murine for angiogenesis testing. Mild ischemia approaches and severe ischemia approaches are present. Occlusion levels variations normally incorporate iliac ligation, ligation femoral, under the branches, ligation femoral with branches excision, vein stripping and dependent variation approach (Goto, 2006 1646) and (Masaki, 2002 1648). Variation in this model kind leads in differing ischemia’s pattern as well as reperfusion (Hellingman, 2010 1649).

            In this study, we adopted the model described by Couffinhal et al. (Couffinhal, 1998 928) that Lofti et al. (2013 p.1657) recognized as the most suitable method for ischemia’s induction in signs production in the mouse similar to the observed and analyzed one in Human CLI. The model is made of distal external iliac ligation as well as femoral and middle segment excision. For reproducibility of the model and since we adopted C57BL/6 mice, with an extensive pre-existing collateral circulation, we also opted for concomitant ligation of the femoral vein to increase ischemia severity (Hellingman, 2010 1649). One of the reasons to choose C57BL/6 mice, which is a largely congenital strain that holds nominal heterogeneity genetic amid animals was the quest of avoiding variability amid mice as it would guarantee reproducible and reliable experiments with the use of similar strains (Bentzon, 2010 1658). When equated to the rest of mice strains. Such as BALB/c and A/J it holds a more comprehensive recovery of perfusion of limb post-ischemia induction and an increased capacity for collateral arteries recruitment (Helisch, 2006 1659) and (Scholz, 2002 1660). Thus this results in the avoidance of hind-limb necrosis thus permitting long-run mice analysis.

            Despite the clinical significance, held by HLIM has clinical value its limitatoons cannot be ignored when equated to CLI our HLIM is acute in nature. Despite the fact that a three-year-old mouse is similar to 80 years human it is more suitable for efficiency to utilize older mice in modeling CLI.  In this context, we utilized 26 weeks old female mice under the C57BL/6 category (Austad, 2009 1662). The husbandry costs that are associated with the use of older mouse results in higher experimental expenses. This is given that older mice are characterized by slower recovery levels after the conduction of femoral artery ligation when equated with younger mice and are lower expected to comprehensively recover without the provision of any therapeutic engagement. Young adult mice which are about three months old holds a 50 percent better functional recovery state when compared with the aged ones which are 18 months 2 weeks after HLI induction (Westvik, 2009 1665). Arteriogenesis seems to be diminished in an ischemic hindlimb for older mice thus revascularization occurs principally via angiogenesis which is less efficient in the current model. An additional limitation of the utilization of younger mice it that they are usually the wild kind and lacks any of the necessary co-morbidities that are commonly observed in CLI patients like hypertension, DM and hypercholesterolemia all linked to slow recovery from ischemia that associates with lower densities capillary.

Interestingly, in patients with PAD, preeminent VEGF plasma was decreased to the normal standard on revascularization. (Makin, 2003 367) and (Pourageaud, 1998 368) Suggested that angiogenesis is enthused during Ischemia of the Limb. An often consequence of these health conditions is inflammation induction as well as stress oxidative resulting in endothelia dysfunction as well as impaired arteriogenesis partially via a reduction both in mediated dilation flow and exterior vascular restructuring (Ziegler, 2010 1666).

Nevertheless, with our murine HLIM, we showed that LDIR provides a kinetic advantage, synergizing with HLI and inducing faster recovery from ischemia with significant improvements in perfusion, capillary, and collateral densities, tilting the angiogenic balance towards an, even more, pro-angiogenic phenotype. In contrast, no effects of LDIR were documented in the resting vasculature, not subjected to ischemia. All the concerns involving inflammation and endothelial dysfunction after ischemia and LDIR were also addressed during our research work .In our mouse HLIM, the acute ischemic stimulus per se, created by the femoral ligation and excision, and triggers a regeneration response by itself (Couffinhal, 1998 928). Mice were expected to regenerate when subjected to ischemia, improving their hind limb perfusion after an abrupt interruption of blood flow supply, namely through capillary and collateral vessels growth.   

Laser Doppler Perfusion Imaging (LDPI) is often utilized in the biomedical study for the evaluation as well as monitoring of mouse microcirculation HLIM for researching in reference to PAD. This was or option for LDIR effectiveness evaluation as the pro-angiogenic intervention in regard to HLI.  This approaches results in minimized pain as well as distress to mice, lowers the animals number that is necessitated in protocols regarding biomedical and permits non-terminal, chronic assessment of superficial physiological mice perfusion.

Subsequently, surgical initiation of autonomous HLI, twenty six weeks old female C57BL/6 rats were fake- exposed or exposed with four diurnal fractions of 0.3 Gy, in uninterrupted days and permitted to recuperate. Femoral artery unilateral excision permitted the Contralateral Limb to be utilized as the comparator for control despite the changes in recruitment or gait of the spinal reflexes mean that the muscle contralateral is not equal to the unprocessed controls (Egginton, 1999 1667)and (Hudlicka, 2003 1668).

Serial perfusion measurements, ensuring similar environmental conditions and mice activity status, were performed before ischemia, immediately after ischemia at day 7, 15 and 45 post-ischemia induction. A drastic blood flow reduction was observed in ischemic limb directly after the surgical procedure when equated to contralateral limb and as per the anticipation, a gradual perfusion improvement was observed over time passage. Strikingly, a significant improvement in blood flow recovery was seen in the LDIR group, 15 days post-HLI that persisted at 45 days post-HLI, compared with control mice. This demonstrates a benefit of LDIR in perfusion recovery, in the setting of HLI (P<0.001). Even though the advances in radiobiology during the past two decades challenge the validity of the LNT theory suggesting that it overestimates radiation risks (Tubiana, 2009 932), or the more recent hormesis hypothesis, the next step in our work, keeping in mind translation to human medicine, was to determine the lowest dose of IR able to induce perfusion recovery.

The utilization of the same exemplary mice were exposed with: (i) inferior number of portions (1* 0.3 GY; 2 x 0.3 GY; or 3 x 0.3 GY); (ii) inferior dose per portion (4 x 0.1 GY); or (iii) higher quantity of portions (7 x 0.3 GY) following ischemia initiation. Non- exposed (NIR) mice were used as a control. No noteworthy enhancements were perceived in muscles exposed with a subordinate number of portions (1 * 0.3 Gy; 2 x 0.3 Gy; or 3 x 0.3 Gy) or with lower dose for each portion (4 x 0.1 GY) when equated to sham-exposed muscles. However, we found that blood perfusion recovery was significantly increased at days 15 and 45 post-ischemia in the ischemic limb of irradiated mice with a higher number of fractions (7 x 0.3 GY), when compared to sham-irradiated mice. Therefore, our results suggest that temporal dynamics for blood flow recovery; angiogenesis or arteriogenesis may be modulated by different doses of IR. These observations accord with previous findings that different doses of IR may conduct to different biological effects since they may differently modulate intracellular signaling (Sofia Vala, 2010 938).

Calling upon the same HLIM, histological and molecular analysis of tissue samples was the next step of our research. Although this evaluation required the sacrifice of the animal’s assessment of capillary density in the gastrocnemius muscle was implemented at several time points. Nevertheless, results are shown at the 15-day post-ischemia and importantly at day 45-post ischemia, after histological complete muscle regeneration. Capillary capacity, for instance, the number of capillaries that exists for every fiber muscle was thus quantified by the staining muscle parts for the endothelial marker in the two differentiated section of four different anatomic sections of every specimen. As expected, HLI per se is associated with a non-significant higher capillary density. However, when comparing irradiated and non-irradiated ischemic gastrocnemius muscles at day 45-post ischemia, there was a significant increase in capillary density (P<0.001) again suggesting a synergy between LDIR and HLI.

According to, Springer, (2000 940) Collateral development denotes the extensive development of the pre-existing vessels and thus it becomes of the highest significance in the recovery of ischemic tissues. Thus, exposing the mice to LDIR (0.3 Gy) or sham-irradiation, during 4 consecutive days after ischemia induction, we were aiming to evaluate if mice would regenerate with higher collateral vessel density (CVD). Through the injection of a contrast agent and the use of the diaphonization technique, we were able to visualize in detail the arterial branching and quantify the CVD. Diaphonization is an ancient preparation anatomical technique that is utilized in making the specimen more transparent with the capability of revealing the three-dimensional arterial distribution of those specimens that have been preserved. In the last century it has been utilized mainly in the study of vascularization in reference to a number of structures and organs (Tilotta, 2009 943), (Bourdelat, 1989 944) and (Alberti, 1989 945). This technique is based on principles of optical physics and involves sequential chemical processing to replace all interstitial fluid by a specific solution, giving a homogeneous density to all tissues. Therefore, through an optimization of optical physical properties, light is transmitted through the thickness of the mouse limb without being reflected neither absorbed, giving it transparency.

Through the injection of an opaque contrast agent before this technique, we are able to perfectly recognize the vascular tree and to quantify the collateral vessels. The region of interest (ROI) was selected in every mouse in equivalent anatomic regions, surrounding the ligation site at the thigh and equivalent to 20% of the overall limb part. These regions represent the best location to measure collateral vessel growth in response to ischemia since it was reported that arteriogenesis occurs primarily around the occluded vessel segment (Zhuang, 2006 948). In the present work, we have shown that at 15 days and at 90 days post-ischemia LDIR promotes collateral vessels growth in the adductor muscles of the thigh (P<0.001) when comparing ischemic irradiated muscles to sham irradiated ones. After establishing that LDIR acts synergistically with HLI inducing a faster recovery from ischemia the next step in our research was to determine the mechanism(s) responsible for this kinetic cooperation.

In order to evaluate the expression of angiogenic genes, using laser microdissection microscope CD31 positive cells were isolated from ischemic, low-dose irradiated and sham-irradiated gastrocnemius muscles. First, assessing the expression levels of endothelial-specific transcripts (platelet and endothelial cell adhesion molecule 1 (Pecam1) encoding CD31, the ETS-related gene (Erg) and ETS variant 2 (Etv2) we confirmed that these CD 31 positive cells were primarily ECs with negligible amounts of myeloid or perivascular cells. According to our in vitro results, several proangiogenic targets (VEGFR1, VEGFR2, FGF2, TGFβ, ANGPT2, PDGFC, HGF and c-MET) were shown overexpressed exclusively in the ischemic irradiated gastrocnemius muscles when compared to the sham-irradiated controls. These findings imply a link for the long-term advantage of irradiated ECs in blood perfusion, capillary density, and collaterals in HLI. LDIR induced a sustained and prolonged pro-angiogenic response in ECs, still evident 45 days after irradiation. Because this contrasts with the transient in vitro response, one may hypothesize either that endothelium itself could be differently modulated by LDIR in a hypoxic microenvironment created by ischemia, or that some cells (e.g. adipocytes) could contribute to perpetuating the effect(s) of irradiation in ways that in vitro cultures cannot mimic. Importantly, no effect was observed in the adductor muscles capillary density or gene expression profile neither in response to ischemia per se, or after LDIR exposure.

Moreover, we showed that the increase in capillary density and the upregulation in relative gene expression found in irradiated gastrocnemius muscle is abrogated or inhibited by treatment with PTK/ZK, a VEGFR2 tyrosine kinase inhibitor. PTK/ZK has no effect on the increase of gastrocnemius capillary density modulated exclusively by HLI. This is in accordance with the fact that blockage of VEGFR2 activation prevents the IR-mediated angiogenic response, as previously reported (Shalaby, 1997 1644). Conversely, the collateral vessel density induced after LDIR exposure was not affected by PTK/ZK. These results suggest that LDIR induces capillary density and pro-angiogenic gene expression in a mechanism dependent of VEGFR2 activation, but this same mechanism do not explain arteriogenesis. EPCs capability to ischemic injuries repair necessitates them to be mobilized initially from BM in the quest of migrating into the Ischemic region where EPCs can thus differentiate into ECs nature. According to our results, LDIR appear to act again synergistically with HLI promoting an earlier and greater increase in the number of serum EPCs identified by flow cytometry as VEGFR2/Sca1/CD117 positive mononuclear cells.

Several cytokines were reported as being involved in the guidance of EPCs to ischemic tissue (Luttun, 2002 1604), (Sennikov, 2002 2042) and (Asahara, 1999 2043). Our previous lab results showed that in hypoxic mimicking conditions LDIR increase ECs VEGF expression (Sofia Vala, 2010 938). In this work we also showed, not only in vitro but also in vivo, that Pgf and Csf3 expression is up regulated in ECs and a significant increase in plasma levels of VEGF, PIGF and G-CSF was observed at day 4 after 4 x 0.3 Gy LDIR exposures. The up regulation of these cytokines, not only VEGF, might explain why PB EPCs levels are not modulated by PTK/ZK inhibition. Our results showed that PB levels of SDF1 (an important cytokine for EPC mobilisation) are not modulated by LDIR (0.3 Gy), contrary to the work of Lerman et al (Lerman, 2010 2056)which showed that a single dose of 5 Gy IR up-regulates SDF-1 through both HIF-1-dependent and independent pathways. Again our results suggest that the effects of IR are dose-dependent.

In order to evaluate the functional relevance of these LDIR-induced circulating EPCs we used a transplantation model, in which all hematopoietic cells were GFP+ (including circulating and extravasated/tissue, leucocytes, erythrocytes, and platelets), but EPCs, forming the inner lining of blood vessels, exhibited both green (GFP+) and red (CD31+). A quantitative evaluation of the histological sections revealed a significant increased number of GFP+/CD31+ cells per area into irradiated thigh muscles when compared to the non-irradiated ones. LDIR per se do not increase these growth factors concentrations and notably the irradiation of the ischemic tissue is critical for the mobilisation of EPCs and collateral formation. Although we cannot exclude that other cells could modulate the levels of these cytokines upon LDIR, our results strongly suggest the involvement of ECs.

The affiliation amongst IR and the resistant system is intricate and multifactorial and is habituated by the prescription and superiority of the emission as well as the sort of cell premeditated. The impacts of the utilization of low doses for the vasculature is that they cannot be extrapolated and are non-linear when equated to higher doses. Irradiation of high doses involves both harmful and protective impacts but doses that are particularly low (0.025 up to 0.05 GY) provided at the reduced dose rate were demonstrated as protective (Mitchel, 2011 2060). According Rodel, (2002 215) DNA lesions induced by IR may be not that severe as to allow greater efficiency of DNA repair mechanisms and epigenetic pathways such as DNA methylation (Aarts, 2011 1908) and the differential expression of several types of proteins may explain the different biological effects of IR (Rodel, 2012 217). In general doses higher than 2 Gy produce pro-inflammatory effects (Williams, 2003 205). On the other hand, doses lower than 1 Gy are responsible for the modulation of several inflammatory processes resulting in unequivocal anti-inflammatory properties. (Rodel, 2007 206)The usefulness of radiation therapy, based on the anti-inflammatory properties of IR, has been long known. Therapeutic applicability was demonstrated on the inflammatory disease as symptomatic improvement of rheumatoid arthritis was observed when mice were irradiated with 0.5 Gy in five fractions within one week (Frey, 2009 2044). Accordingly symptomatic improvement was additionally shown in a mice model or arthritis that had been induced with irradiation doses of a single Gy with 5 fractions and 0.5 Gy with 5 fractions, in a mechanism dependent on reduced Inducible Nitric Oxide Synthase (iNOS) activity and increased heme oxygenase  (HO-1) levels (Hildebrandt, 2003 2058).

Hematopoietic infiltrate in the study’s HLIM was supervised from reddened and ischemic tissues to evaluate the probable responsibility of resistant cells upon LDIR. Ischemia per se induced about a 20 times to increase in the immune CD45+ cell infiltrate recruited to the injured muscle. Exposure with LDIR significantly inhibited the CD45+ cell accumulation with particular effects on monocytes, macrophages, and neutrophils. When 2.0 Gy were administered during 4 consecutive days the total CD45+ accumulation in ischemic muscle was still reduced as compared to sham-irradiated mice, although numbers of monocytes and macrophages were restored, not neutrophils. This is consistent with the fact that high irradiation doses have opposing effects on certain myeloid subsets, for they activate macrophages (Klug, 2013 2050) while they are reported to induce rapid, but transient, neutropenia (Romero-Weaver, 2013 2051).

            Importantly, the effect of irradiation was short-listed. Fifteen days post-HLI the profiles of myeloid cells that infiltrated non-irradiated and irradiated ischemic muscles were similar. Altogether these data pointed for a mechanism of LDIR-induced angiogenesis independent of local myeloid cell recruitment and suggest like previously mentioned, that LDIR may even have anti-inflammatory proprieties. One important concern when addressing IR is its toxic effect. According to the currently challenged LNT hypothesis, the dose-response is linear and no threshold exists where damage begins to show. Higher doses of IR (2 to 10 Gy) have been used to induce revascularization in several studies (Heissig, 2005 933), (Thanik, 2010 934) and (Zhou, 2009 936) and in fact radiation therapy still continues to be an accepted treatment for benign diseases (Jha, 2008 937). However, there is no consensus about the doses described as pro-angiogenic, as different radiation sources are used. The use of conventional radiotherapy dose (2-10 Gy, administered once, Caesium-137 source) has been shown to induce neovascularization in HLI via the release of VEGF from the mast cells as well MMP-9 progenitor mediated mobilization cells. However, some probably advanced implications were referred (Heissig, 2005 933). To the best of our knowledge, to date, the use of those high doses has not been proposed for therapeutic angiogenesis.

Here, IR was delivered through a linear accelerator producing x-rays photon beam, currently used in the clinical practice. In order to assess the long-term toxicity of LDIR, we used the same HLI model and LDIR regimen described previously. No difference was seen in weight gain (at weeks 24, 36, 48 and 52 post-HLI) between control (sham-irradiated) and LDIR animals. At week 52 post-HLI mice were sacrificed and morphological, biochemical and histological parameters recorded. No morbidity or mortality was observed. Urinalysis and blood count, serum biochemistry and coagulation tests results were within normal range for the C57BL/6 mouse strain, for both groups. Bone marrow smear and histological sections of skin, lung, spleen, muscle, bone marrow and lymph nodes were analyzed and the few lesions identified were common to all experimental groups, mostly related to aging and the strain of the mice used (Haines, 2001 2052), and not associated with LDIR. Thus far, and although the possibility of LDIR long-term toxicity cannot be completely ruled out, as genomic instability and non-targeted bystander effects are delayed effects of IR, no LDIR-associated toxicological effects were observed. Furthermore, the beneficial effects of a therapy in a disease with limited life expectancy, as CLI, can overcome its putative negligible or minor toxic effect.

In other words, using a model of experimentally induced HLI, mice were exposed to LDIR and limb perfusion, capillary density and collateral vessel formation were measured. We show that LDIR (4 x 0.3 Gy) improve limb perfusion by enhancing arteriogenesis through EPCs recruitment to sites of collateral vessel development, an effect dependent on exposure of the ischemic niche to LDIR, but not on the local recruitment of myeloid cells. Likewise, LDIR also favors angiogenesis through simultaneous activation of a repertoire of pro-angiogenic factors in mature ECs in a mechanism dependent on VEGFR signaling, with no short-term side effects and no effects on resting vasculature, opening a possibility to new therapeutic strategies in lower limb vascular insufficiency.

CELL THERAPY

Vasculogenesis incorporates the recruitment of Progenitor cells towards the ischemic region as well as their differentiation in the fresh blood vessels. The most suitable cell for the cellular therapy in regard to CLI which has retained its controversy till today. Most of the potential cells candidates have thus been assessed as neovascularization promoters with the inclusion of BM cells, Hematopoietic stemming cells, BM-MNCs, EPCs, MSCs, and Hemangiocytes. MSCS can be regarded as multi- powerful non-hematopoietic cells like fibroblast that can be excluded from the differing tissues which incorporate BM, placenta, adipose tissue and blood in the umbilical cord.  Normally therapeutic vascular effects are exerted by MSCs through paracrine factors secretion that might incorporate anti-inflammatory as we as immunomodulatory implications. Immune-modulation is a unique feature for MSCs, making their combination with other stem cells subtypes very appealing to enhance allogeneic injection for inducing repair.

Studies have demonstrated that cells acquired from adult patients with numerous risk forces hold compromised functions. Based on this rationale, therapeutic efficiency for CLI patients can be raised by the use of MSCs acquired from younger donors overcoming age and vascular disease–mediated deficiencies in EPCs number, function, and angiogenic cytokine production. Amongst the probable MSCs sources, there lies the umbilical cord tissue. Stem Mesenchymal Cells acquired from the umbilical cord Tissues (UC-MSCs) hold equated surface phenotype, multipotency, and plastic adherence similar to MSCs derived from different sources. Compared with the differing parts from other regions, UC-MSCs have acquired undoubtedly benefits:

1. To begin with, they are associated with a non-aggressive gathering procedure for allogenic or autologous use which is acceptable ethically.
2. Hold a reduced infection risk
3. Reduced teratoma risk
4. Easy to acquire a number that is considered for UC-MSCs post a number of passages and excessive Ex-vivo rise.
5. Rapid self-renewal when equated to BM-MSCs
6. Hold low immunogenicity that has the desirable immunosuppressive capability (Nagamura-Inoue, 2014 2040).

ECBio, one of the leading biotechnology corporations, has created a proprietary advancement to constantly isolate, increase and cryopreserve a population that is suitably organized into the human cells stem acquired from tissues within the umbilical cord by the term Wharton that has been titled UCX® cells (Santos, 2013 1955). The utilized technology for UCX® segregation differs from all other UC-MSCs segregation methodologies. In short, the intuition of the three prime stage approach for the recovery of cells was intuited in the quest of making the approach completely reliable.  Second, the segregation starts with amniotic membrane peeling off with a reduction of microbial contamination frequency thus augmenting the accuracy of UCX® resulting populace by the general epithelial progenitors’ elimination. Third, tissues incisions or the crushing criteria is avoided given that it would hinder its use in proper manufacturing operations setting. In addition, it is likely to jeopardize phenotype stem cell based on the presence of extreme technical manipulation. Furthermore, the utilization of optimized ration amid mass tissues, general solution capacity, enzymes operative units digestion and empty capacity UCX® particular cells release thus inducing vessels cord that will, in turn, lower the endothelial contamination and cells sub-endothelia from umbilical vein and arteries (Santos, 2013 1955).

UCX® cells can be termed as a populace that is standardized belonging to stem cells that adhere to the existing  MSCs description as recognized mainly by the (ISCT)- International Society for Cellular Therapy (Santos, 2013 1955) and (Gartner, 2014 2033). Notably, it was demonstrated that UCX® cells might turn to an effective and guaranteed fresh approach for the treatment of both systematic as well as local inflammatory arthritis manifestations.  Based on the observations UCX® cells hold the density to inhibiting the human T- cell concomitantly and proliferation thus promoting Tregs expansion efficiency higher than that of BM-MSCs. In accordance, UCX® cells xenogeneic administration in both chronics induced adjuvant and acute induced-carrageenan arthritis models for inflammation arthritis can lower Edema Paw in vivo with increased efficiency as compared to BM-MSCs and demonstrated higher systematic as well as local remission arthritic manifestation. It is authoritatively stated by this information that UCX® cells exist as immunosuppressive and additionally hold lower immunogenicity that is appropriate (Santos, 2013 1955).

Furthermore, it was demonstrated that UCX® cells conserves cardiac functioning and offset adversative remodeling muscle post intramyocardial replacement in the myocardial murine infarction approach. The protective- cardio UCX® effects are exerted via a paracrine approach that appears in the quest of angiogenesis enhancement. In this case, it was demonstrated that UCX® cells raise the capillary volume in regard to myocardium infarcted given that CD31 expressive cells proved to be highly abundant within the UCX® cells transplanted heart wall when equated to hearts injected vehicle. UCX® cells proved to be responsible in quickening wound remedial. The studies suggest strongly that UCX® cells normally act in differentiated cells kinds via paracrine models. Furthermore, the results showed that UCX® Vitro conditioned medium triggers angiogenesis through promoting capillary-like formations by HUVECs (Santos, 2013 1955).

It was recognized that UCX®, when assimilated with HUVECs, arouses tubule HUVECs establishment to the uppermost degree as it is known to be FGF2 pro-angiogenic which is further involved in movement elevation. As demonstrated earlier, for the earlier cells kinds, our findings suggest that UCX® normally serves in ECs via paracrine machinery because a critical rise for the pro-angiogenic forces like PDGFAA, HGF and IL8 were established in the medium UCX® condition while being equated to that of control. In accordance, the findings have substantiated the findings demonstrating that UCX® ECS, and tubulogenesis migration.

In the quest of investigating whether UCX® was capable of therapeutic angiogenesis promotion in the context of HLI, the study utilized once more the26 week’s old female mice. Post the surgical unilateral HLI induction two differentiated UCX® were assessed: intramuscular in the gastrocnemius muscle, 5 hours post-ischemia and retro-orbital (RO), 24 hours post-ischemia induction. The involved risk in rejection of immune in this study’s experimental set up was accounted as minimal given that UCX® had been xenon- entrenched prior into immunocompetent rabbits, sheep,  mice as well as rats without an elicitation of any compromising rejection of immune (Santos, 2013 1955) and (Santos Nascimento, 2014 1956).

Different works have shown that the delay between the onset of disease and the administration of MSCs could affect the therapeutic efficacy (Yan, 2013 2031). In contrast, using similar models, other trials show that MSCs are more effective when administered 24 hours after ischemia induction (Prather, 2009 2032). In our study, mice died after 5x106 UCX® administration via RO injection, the highest cell concentration causing lung embolization. A lower cell concentration (1x106) was tolerated, but UCX® were not visualized in the gastrocnemius muscle after 2, 7 or 15 days, as assessed by immunofluorescence microscopy. Intra-muscular injection has been the utmost frequently developed technique both in pre-medical and clinical studies even though the needle can cause tissue damage (Yan, 2013 2031). Therefore, 2x105 or 1x106 UCX® were injected via IM, 5 hours after ischemia. At day 7 post-ischemia induction, UCX® were present in the gastrocnemius muscle as assessed by immunofluorescence microscopy, but the concentration of cells was decreased when compared to day 2. Moreover, at day 15, UCX® was no longer detected. We may hypothesize that following UCX® administration, cells survive a few days due to their already described low immunogenicity and then die.

Human MSCs has been labeled as niche and home to inflammatory locations retaining their viability in Xeno-relocated rat for about two weeks in standard rats/ mice and thus raising their inflammation sites permanence in diseased approaches without noticeable coming to the differing organs (Gartner, 2014 2033). In another study, it was shown that when 1x106 placental human MSCs expressing luciferase were additionally intuited and it was demonstrated by the bio-pattern distribution that the capability of cells persistence only occurred at the site of injection and was not distribute to the differentiated organs. Additionally, placental MSCs for humans sustained constant high luciferase levels expressive cells for approximately three weeks (Gartner, 2014 2033). In order to follow UCX® cells over time, biodistribution analysis was performed; however, we show that the number of administered luciferase-expressing cells (2x105 cells) is not enough to allow their detection by IVIS Lumina, even immediately after IM injection. For that reason, it was not possible to follow up the UCX® residence in the gastrocnemius muscle over time. Moreover, we cannot assure that UCX® does not migrate to other surrounding organs/tissues or enter into the bloodstream. For that motive, another technical approach was used in the mandate to scrutinize the supposed presence of UCX® not only in the gastrocnemius muscle but also in other organs such as lung, spleen, liver, and kidney. Accordingly, these organs were isolated from ischemic mice sacrificed at days 3 and 28 post- UCX® injection and RT-PCR will be performed using a human gene target. According to our results from immunofluorescence, UCX® should be detected in the gastrocnemius muscle at day 2. Given the high sensitivity of the RT-PCR method, it would be important to know if UCX® remains at the gastrocnemius muscle (injection site) at day 28. Simultaneously, and using the same approach, we expect to evaluate the capacity of UCX® to migrate to and proliferate in other organs.

With the objective of assuring the safety of UCX® cells, another route of UCX® administration was used in our biodistribution analysis. Therefore, after ischemia induction and UCX® intraperitoneal (IP) administration, cells were detected proximal to femoral artery excision site at day 0 or 1 post-UCX® injection. These results were in accordance with the literature. However, in one of these mice, a strong signal was also detected, at days 0 and 1, in the abdominal area (Sensebe, 2013 2035). We may hypothesize that this particular mouse had an inflammation/injury at the abdominal area when ischemia was performed/ UCX® administered and for that reason, UCX® cells were driven to that area. It is also possible to hypothesize that UCX® had a secondary homing; however, no signal was detected in the lung. This specific mouse was sacrificed at day 1 post-UCX® injection in order to analyze if the abdominal area presented any inflammation and/or injury and track the cells by RT- PCR. The other two mice of the same experimental condition were followed over time and they were sacrificed at days 7 and 15 post-ischemia, when both presented an absence of bioluminescence signal, in order to track UCX® by RT-PCR in the principal organs (ex: gastrocnemius muscle, lung, spleen, liver, kidney). A similar experiment was performed using non-ischemic mice. In this experimental condition, UCX® were not detected by bioluminescence analysis after IP injection at any time point. With the same objective, mice were sacrificed at days 7 and 15 in order to track UCX® by RT-PCR in the principal organs.

After establishing the safety of IM injections as the best administration route and the 2x105 the best dose, the next step in our work was to assess if UCX® promotes angiogenesis and arteriogenesis in this mouse model of HLI. Accordingly, perfusion recovery was evaluated over time, capillary density was measured in the gastrocnemius muscle and collateral density was measured in the adductor muscle. Our results showed an increment in perfusion recovery in mice treated with 2x105 UCX® as assessed by laser Doppler at 14, 19 and 24 days post-ischemia when compared to untreated ones. Moreover, an increase in capillary density and collateral vessel density (CVD) was noted in mice treated with 2x105 UCX®.

            These assessments were conducted mainly on the 90th day after HLI creating the suggestions that the implication that was persuaded by UCX® in both arteriogenesis and angiogenesis is sustained over a time period. Together with, the on-ischemic forces are not impacted by UCX® gave that capillary capacity as well as CVD is equivalent to the muscles that are on-ischemic both derived from control mice as well as UCX® treated ones creating the indication that UCX® might generate a resident action. The data thus suggest that UCX® holds the capability of secreting cytokines and practically alter ischemic mice surrounding thus contributing towards therapeutic angiogenesis.

            Moreover, in this work, the in vivo angiogenic potential of UCX® obtained from two different umbilical cord tissues was compared. The cells, isolated from two different umbilical cords, presented differently in vitro angiogenic potential according to ECBio’s results (secretome analysis and matrigel tube formation assays). According to our in vivo results, at days 14 and 21 post-ischemia we found that mice treated with either UCX® present similar levels of blood flow, both significantly higher when compared to untreated mice (p˂0.01). However, at day 7 post-ischemia, the perfusion recovery was significantly increased only in mice treated with UCX® presenting the best angiogenic potential in vitro. Our results suggest that UCX®, isolated from the tissues of different umbilical cords and presenting differently in vitro angiogenic potential could induce perfusion recovery after HLI with different temporal dynamics. It will certainly be important: 

1. To establish through analysis if those UCX® have the same potential in inducing capillary and collateral densities
2. To gather knowledge about the molecular and cellular mechanisms by which those UCX® induce perfusion recovery and consequently capillary and/or collateral densities. According to the secretome analysis (performed by ECBio), those UCX® secrete the same cytokines/chemokines but their levels are different. We may hypothesize that those factors (including pro-angiogenic factors) being present in different levels will interfere differently with the microenvironment and modulate in a different way the intracellular signaling of different target cells. This could contribute to different biological responses and/or same biological responses at different levels. It is important to remember that the angiogenic switch has to occur to initiate angiogenesis and a dynamic balance between pro- and anti-angiogenic factors control this process.

            It should be noted that a fresh UCX® mechanical action is demonstrated for the initial time. UCX® moderates pro-angiogenic expression players within ECs. The study established that 70 days after UCX® and HLI treatment, ECs segregated from UCX® micro-surrounding treatment and presented an upward parameter in their expressive rate in regard to HGF, FGF2, Ang2, and TGβ2 after being segregated from the ischemic muscle gastrocnemius when equated to the contralateral ones. This implication is UCX® specific given that the contrary one is verified within the mice control. Based on these findings it can be established that UCX® concurrently regulates several antigenic ECs forces. As per the current mechanism, the hypothesis is then that UCX® advances angiogenic endogenous effectiveness in respect to ischemic framework.  Keeping in mind translation to human medicine, another very important topic of this investigation was to assess if cryopreservation and subsequent thawing, both in vitro and in vivo, did not impair phenotype, immunomodulatory and angiogenic potencies of this specific UC-MSC population. Even though a noteworthy effort has been expended in order to optimize the manufacturing process to guarantee that MSCs retain their safety and therapeutic proprieties the results remain controversial.

            Reports demonstrate that cryopreservation negatively affects the immunosuppressive properties of BM-MSCs in a reversible manner, and is associated with a heat-shock stress response initiated during the thawing process, triggering the instant blood-mediated inflammatory reaction, occasioning to quicker complement- arbitrated removal next to blood acquaintance (Moll, 2014 1964). Temporary repression of non-constitutively expressed genes during the stress response allows the cells to prioritize cell survival before recovering their functional properties (Francois, 2012 1963). MSCs administration has been demonstrated to having the capability to improving renal functionality in rodent replicas of chronic kidney sickness partially by the reduction of intrarenal suppression and inflammation-fibrosis. Nevertheless, similar claims have been reported concerning the administration of cryopreserved adipose tissue-derived MSCs (AT-MSCs) re-counting noteworthy opposing implications and no apparent clinically applicable enhancement in renal efficient limits. AT-MSCs administration refined from adipose cryopreserved was not linked to opposing implications but was not additionally linked to renal functionality limits improvement (Quimby, 2013 1966).

            However, the belief that cryopreservation negatively affects the performance of MSCs has recently been challenged. Lützkendorf et al. showed that xenon-free, GMP-grade BM-MSCs, cryopreservation had no impact on viability and consensus criteria of MSCs. In co-culture with PB-MNCs, MSCs showed low immunogenicity and suppressed mitogen-stimulated proliferation of PB-MNCs irrespective of cryopreservation. Cytogenetic aberrations were not observed consistently in fresh and cryopreserved products, and no signs of malignant transformation occurred in functional assays (Luetzkendorf, 2015 1967). More recently, Cruz et al. in vivo analysis verified the potent xenogeneic effects of human BM-MSCs in an immunocompetent mouse model of allergic airways inflammation and established that thawed MSCs are as effective as fresh MSCs (Cruz, 2015 1968). Most importantly, in phase II clinical study using BM-MSCs in acute graft-versus-host disease in which either cultured or freshly thawed cell was infused, there was no report of clinical differences observed between the two groups (Le Blanc, 2008 2075).

            The paradoxes between reports, some suggesting that freshly thawed previously cryopreserved MSCs may not have the same effectiveness or breadth of anti-inflammatory activities as do continuously cultured MSCs may be explained by differences in tissue source and manipulation during the manufacturing process, including the cryopreservation and thawing procedures (Barcia, 2015 1971). Previous studies used BM and AT-derived MSCs, conducted by Francois, (2012) and Quimby, (2013) whereas the present work reports to UC-MSCs. It was demonstrated by the findings that UCX® proved to possess low immunogenic and higher immunosuppression operation when equated to BM-MSCs. More, UCX® did not necessitate activation before or exert priming to their immune-modulatory implications.

            As mentioned before, Francois et al. (Francois, 2012 1963) in their work concluded that cryopreserved BM-MSC that are freshly liquefied acquired from standard human volunteers that regulated protein’s heat shock are headstrong to interferon. (IFN)-γ-prompted up-parameter of IDO, and cooperate in overwhelming CD3/CD28-single-minded T cell propagation. On the contrary, this works findings show that cryopreservation has no deleterious effect on the immune-modulatory potential of non-primed UC-MSCs, as seen in vitro by the proliferation suppression of PB-MNCs activated by Non-CD3, Non-CD28 as well as IL-2, and also no differences were documented in the expression of some functional markers CD200, CD274, CD273 and CD146 by cryopreservation as a whole. Although the immune functionality testing in vitro of UCX® cells, as performed in this study, was only partial, and the impact on cell biochemical responsiveness to inflammatory cues such as interferon-γ, tumor necrosis factor-α, IL-1 or toll-like receptor agonists were not evaluated, our results support the thesis that UC-MSC immunosuppression activity is independent of priming mechanisms (Santos Nascimento, 2014 1956) and (Miranda, 2015 1957) and that it is not affected by cryopreservation.

            Having established that freshly thawed UCX® maintained their functional markers and their ability to suppress activated T cells, we further tested whether freshly thawed UCX® cells showed impaired immune-modulatory therapeutic benefits in vivo. For this purpose, a prolonged adjuvant -induced arthritis (AIA) rat ideal was utilized over the course of 64 days. The results showed that there was no significant difference between treatments performed with either cultured or freshly thawed cell for any readout measured. In this study, we obtained no evidence of increased apoptosis or changes in surface markers in the freshly thawed population compared with the cultured population, and although clearance and engraftment were not directly compared in vivo, the two conditions showed no differences in therapeutic activity/potency in vivo.

            Regarding angiogenic potential, the previous results were obtained using UCX® cultured during five days before the administration in ischemic mice. In addition, in this work we also compared the effect obtained in perfusion recovery of UCX® in these conditions with: i) UCX® administered immediately after thawing and ii) UCX® administered after 24 hours in culture. Our results suggest that at days 14 and 21 post-ischemia no significant differences were obtained between them. The three different experimental conditions present similar levels of blood flow that are significantly different when compared to the levels of untreated mice. However, at day 7 post-ischemia, UCX® administered after 24 hours in culture did not show significant differences when compared to untreated mice, in contrast to UCX® cultured during five days before the administration or UCX® administered immediately after thawing. In order to explain these results, we may hypothesize that UCX® thawed and placed in culture during 24 hours are not yet adapted to in vitro culture conditions and are not proliferating in contrast to UCX® cultured during 5 days before administration. Therefore, according to the in vitro culture condition, UCX® may present different molecular expression patterns that will differently influence the ischemic microenvironment contributing to a different response in the first few days after administration. After that period of adaptation to an in vivo microenvironment UCX®, that survive could conduct similar biological responses. Concerning the condition where UCX® were administered immediately after thawing, it is possible that the present results are a consequence of the fact that the cells did not have to pass through the adaptation to different conditions in a short time span. This indicates that UCX® cells do not require a recovery period and may be infused immediately after thawing.

            Besides cell source, process-related factors that might have contributed to this outcome concern the drastic reduction of tissue mechanical manipulation of ECBio specific isolation process, also, ECBio process development involved a thorough optimization of the tissue preparation process and digestion parameters that resulted in a neglectful contamination of epithelial, endothelial and sub-endothelial cells, thus promoting the phenotypic homogeneity of the final population.

            Conclusion and Future Perspectives

            In summing up, the preclinical, as well as the clinical tests on cell and gene therapy in referent to the two recent decades, created a principle roof in that angiogenesis therapy can be described as an alternative treatment for patients with no option suffering primarily from the vascular disorder that incorporates CLI. However, the acquired efficiency that is remarkable in reference to angiogenic therapy that was observed in preclinical models, has not yet been translated into clinical trials. Although scientific research has added various weapons to the therapeutic armamentarium in the battle against the cardiovascular ischemic disease, it has also raised a number of issues yet to be solved: optimal gene or cell delivery methods, timing and dosing regimens; and effective cell populations, as well as, isolation and processing methods.

            Low dose ionizing radiation may hopefully become a new tool in regenerative medicine, since they activate, simultaneously, several pro-angiogenic factors and also increase EPCs mobilization improving their function, proliferation and survival features, positioning themselves as an adjuvant therapy that allows overcoming endogenous impairments in EPCs health and vascular responsiveness. These mechanisms could be extremely relevant given the complex and fine-tuned process that represents the formation of a functional vessel network, requiring the constant interplay of several angiogenic factors.

            The mechanisms described in our research allow us to overcome some of the most pressing limitations currently pointed to therapeutic angiogenesis and also to think of LDIR as a way of improving microcirculation in association with macrovascular revascularization. Regarding cell therapy, our research showed that UCX® may be perceived as a suitable therapeutic methodology for CLI. In a significant context, UCX®   induces perfusion of blood, capillary volume and the development of collateral. This would partially be achieved via the development of fresh mechanisms because it was demonstrated that UCX® moderates concurrently the expressive nature of a number of pro-angiogenic forces in ECs that would enhance their angiogenic effectiveness. Again this can be applied when being equated to additional approaches where just one angiogenic force is administered, particularly if angiogenesis is understood as a complex procedure that incorporates a number of cytokines. Concerning the manufacturing procedure, in addition to dispensing the need to histo-compact tissues matching which can be less expensive probably and more convenient considerately more than AT-MSCs and BM.

            Future Perspectives

            This study’s results show that UCX® maintains strong immune-modulatory capacity and angiogenic potential, regardless of cryopreservation, do not require priming or a recovery period after thawing validating them as a viable base technology for the production of allogeneic, off-the-shelf, cryopreserved MSC-based advanced therapy medicinal products (ATMPs) with great therapeutic potential. As a future perspective, since the prevalence of DM is increasing, and since DM is one of the main risk factors for CLI, it would be very important to evaluate the efficacy of LDIR and UCX® in a diabetic mouse HILM as endothelial dysfunction is a main feature of DM. Regarding UCX® and the possibility of EPCs modulation, it would be also very significant, in the field of PAD, to identify which molecules could be used as an adjunctive therapy to improve UCX® angiogenic potency and to understand if UCX® administered with own patients impaired EPCs could act synergistically with them improving their function. In the future, using our mouse HLIM we intend to demonstrate that irradiation of UCX® using LDIR will allow them to acquire a more distinct pro-angiogenic phenotype, improving their effectiveness and efficiency as cellular therapy in the CLI. Finally, the success of our clinical trial will give us more impute regarding LDIR and CLI as an important clinical discovery worldwide, with major impact in contemporary clinical practice.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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