Augusto Ministro

Umbilical cord tissue–derived mesenchymal stromal cells maintain immunomodulatory and angiogenic potencies after cryopreservation and subsequent thawing

BACKGROUND AIMS The effect of cryopreservation on mesenchymal stromal cell (MSC) therapeutic properties has become highly controversial. However, data thus far have indiscriminately involved the assessment of different types of MSCs with distinct production processes. This study assumed that MSC-based products are affected differently depending on the tissue source and manufacturing process and analyzed the effect of cryopreservation on a specific population of umbilical cord tissue-derived MSCs (UC-MSCs), UCX®. METHODS Cell phenotype was assessed by flow cytometry through the evaluation of the expression of relevant surface markers such as CD14, CD19, CD31, CD34, CD44, CD45, CD90, CD105, CD146, CD200, CD273, CD274 and HLA-DR. Immunomodulatory activity was analyzed in vitro through the ability to inhibit activated T cells and in vivo by the ability to reverse the signs of inflammation in an adjuvant-induced arthritis (AIA) model. Angiogenic potential was evaluated in vitro using a human umbilical vein endothelial cell-based angiogenesis assay, and in vivo using a mouse model for hindlimb ischemia. RESULTS Phenotype and immunomodulatory and angiogenic potencies of this specific UC-MSC population were not impaired by cryopreservation and subsequent thawing, both in vitro and in vivo. DISCUSSION This study suggests that potency impairment related to cryopreservation in a given tissue source can be avoided by the production process. The results have positive implications for the development of advanced-therapy medicinal products.

Therapeutic angiogenesis induced by human umbilical cord tissue-derived mesenchymal stromal cells in a murine model of hindlimb ischemia

BackgroundMesenchymal stem cells derived from human umbilical cord tissue, termed UCX®, have the potential to promote a full range of events leading to tissue regeneration and homeostasis. The main goal of this work was to investigate UCX® action in experimentally induced hindlimb ischemia (HLI).MethodsUCX®, obtained by using a proprietary technology developed by ECBio (Amadora, Portugal), were delivered via intramuscular injection to C57BL/6 females after unilateral HLI induction. Perfusion recovery, capillary and collateral density increase were evaluated by laser doppler, CD31 immunohistochemistry and diaphonisation, respectively. The activation state of endothelial cells (ECs) was analysed after EC isolation by laser capture microdissection microscopy followed by RNA extraction, cDNA synthesis and quantitative RT-PCR analysis. The UCX®-conditioned medium was analysed on Gallios flow cytometer. The capacity of UCX® in promoting tubulogenesis and EC migration was assessed by matrigel tubule formation and wound-healing assay, respectively.ResultsWe demonstrated that UCX® enhance angiogenesis in vitro via a paracrine effect. Importantly, after HLI induction, UCX® improve blood perfusion by stimulating angiogenesis and arteriogenesis. This is achieved through a new mechanism in which durable and simultaneous upregulation of transforming growth factor β2, angiopoietin 2, fibroblast growth factor 2, and hepatocyte growth factor, in endothelial cells is induced by UCX®.ConclusionsIn conclusion, our data demonstrate that UCX® improve the angiogenic potency of endothelial cells in the murine ischemic limb suggesting the potential of UCX® as a new therapeutic tool for critical limb ischemia.

Low-dose ionizing radiation induces therapeutic neovascularization in a pre-clinical model of hindlimb ischemia

Aims We have previously shown that low-dose ionizing radiation (LDIR) induces angiogenesis but there is no evidence that it induces neovascularization in the setting of peripheral arterial disease. Here, we investigated the use of LDIR as an innovative and non-invasive strategy to stimulate therapeutic neovascularization using a model of experimentally induced hindlimb ischemia (HLI). Methods and results After surgical induction of unilateral HLI, both hindlimbs of female C57BL/6 mice were sham-irradiated or irradiated with four daily fractions of 0.3 Gy, in consecutive days and allowed to recover. We demonstrate that LDIR, significantly improved blood perfusion in the murine ischemic limb by stimulating neovascularization, as assessed by laser Doppler flow, capillary density, and collateral vessel formation. LDIR significantly increased the circulating levels of VEGF, PlGF, and G-CSF, as well as the number of circulating endothelial progenitor cells (EPCs) mediating their incorporation to ischemic muscles. These effects were dependent upon LDIR exposition on the ischemic niche (thigh and shank regions). In irradiated ischemic muscles, these effects were independent of the recruitment of monocytes and macrophages. Importantly, LDIR induced a durable and simultaneous up-regulation of a repertoire of pro-angiogenic factors and their receptors in endothelial cells (ECs), as evident in ECs isolated from the irradiated gastrocnemius muscles by laser capture microdissection. This specific mechanism was mediated via vascular endothelial growth factor (VEGF) receptor signaling, since VEGF receptor inhibition abrogated the LDIR-mediated gene up-regulation and impeded the increase in capillary density. Finally, the vasculature in an irradiated non-ischemic bed was not affected and after 52 week of LDIR exposure no differences in the incidence of morbidity and mortality were seen. Conclusions These findings disclose an innovative, non-invasive strategy to induce therapeutic neovascularization in a mouse model of HLI, emerging as a novel approach in the treatment of critical limb ischemia patients.

Mycotic Pseudoaneurysm After Kidney Transplantation: Two Case Reports

BACKGROUND Vascular complications after kidney transplantation may cause allograft loss. Here, we describe 2 patients with extrarenal mycotic pseudoaneurysm after kidney transplantation. PATIENTS Patient 1 was a 54-year-old man who developed pseudoaneurysm 60 days after transplantation, and patient 2 was a 48-year-old woman who was diagnosed with a pseudoaneurysm 5 months after transplantation. RESULTS Patient 1 had a deceased-donor kidney transplant with end-to-side external iliac arterial anastomosis that was reconstructed 8 days after transplantation owing to rupture and major bleeding. At 60 days after transplantation, he had high serum creatinine level and Doppler ultrasonography showed a pseudoaneurysm of the arterial graft anastomosis and postanastomotic renal artery stenosis. Treatment included surgical excision of the pseudoaneurysm, vascular reconstruction, and fluconazole, with mycologic culture of the resected pseudoaneurysm showing Candida albicans. Patient 2 developed nondisabling intermittent claudication at 5 months after kidney transplantation, with a pseudoaneurysm subsequently observed on Doppler ultrasonography and computerized tomographic angiography. Treatment included renal artery thrombectomy and common iliac bypass to the hilar donor renal artery with inverted ipsilateral long saphenous vein. Operative samples showed C albicans, and she was treated with fluconazole. Both patients had satisfactory outcomes, and both kidney allografts were preserved. CONCLUSIONS Extrarenal mycotic pseudoaneurysms after kidney transplantation require a high index of suspicion for early diagnosis, and preservation of the kidney graft may be achieved with the use of surgical treatment and antifungal therapy.

Type B aortic dissection in a chronic haemodialysis patient

A 61-year-old African-American patient presented to the emergency room with severe back pain. The patient had a history of end-stage renal disease on haemodialysis for 6 years, chronic obstructive lung disease and uncontrolled hypertension. CT scan revealed type B aortic dissection extending from the left subclavian artery to the right renal artery (figures 1⇓–3). There were no signs or symptoms of malperfusion. The patient was medically managed; blood pressure control required four different classes of antihypertensive drugs. It should be noted that the patient had consistently refused antihypertensive treatment previously. …

Sigmoidization of the hypogastric artery.

A 64-year-old male underwent a routine pelvic ultrasound, during which a conglomerate of vessels was noticed around the prostate and bladder, presenting a flow pattern compatible with multiple arteriovenous fistulas (AVFs). A computed tomography (CT) angiography revealed a huge dilatation of the right hypogastric artery, from its origin to the pelvic floor, with increased diameter and tortuosity in its distal half (A; Cover). Multiple anomalous vessels were noticed around the prostate and bladder (B), related to the nidus of the arteriovenous malformation (AVM). The right common iliac artery was also dilated and tortuous. The diagnosis of extensive pelvic AVM was made, with associated aneurysmatic degeneration of the afferent vessels, the right hypogastric and common iliac arteries. Four years later, the patient presented with symptoms of intestinal subocclusion, requiring a colonoscopy, which proved negative, and a new CT angiography disclosed a remarkable external compression of the rectum by the megaartery. The patient underwent surgical repair, which consisted of resection of the mega-artery from its origin down to the surgically accessible distal level, thus relieving the rectal compression.Theaneurysmatic right common iliac arterywas resected and replaced by prosthesis from the aorta to the external iliac artery (C). The postoperative course was uneventful, and, on follow-up, the patient was found completely free of symptoms. The circulatory pattern of AVFs, either congenital or acquired, has been the subject of multiple studies aimed at the evaluation of their hemodynamic and structural consequences, at the proximal or distal circulatory beds. At the distal level, a significant increase in venous output is noticed, leading in the long run to cardiomegaly or congestive heart failure, sometimes the first clinical manifestation of the malformation. At the proximal level, the dilatation of the afferent vessels has been regarded as a common feature in the congenital and acquired AVFs. The dramatic reduction of peripheral resistance causes significant structural alterations in the arterial wall architecture, leading to its dilatation. In the present case, the arteries became not only enlarged, but also tortuous, resulting in an extraordinary shape, which was fortunately coined by our radiologist as “sigmoidization.”