ClinUCX (2013-2015) – Development of innovative medicines for the treatment of cardiovascular diseases.

QREN/SI I&DT – LISBOA-01-0202-FEDER-30196

Cardiovascular diseases (CVDs), such as peripheral artery disease (PAD) and myocardial infarction (MI), belong to a group of disorders of the heart and blood vessels. CVDs are the first cause of death worldwide. An estimated 17.5 million people died from CVDs in 2012, representing 31% of all global deaths (WHO).

PAD and MI are both conditions provoked by extra cholesterol, and other fats circulating in the blood, that accumulate in the walls of the arteries. This build up, called plaque, narrows the arteries, often reducing or blocking blood flow. In PAD, blood circulation is mainly affected to the limbs, but can also affect the arteries that carry blood from the heart to other organs. Nearly everyone who has PAD have reduced ability to walk and critical cases may evolve to finger or even lower limb amputation. So far there is no curative treatment for PAD.

Given the pro-angiogenic properties of the UCX® product, ECBio has promoted, in collaboration with AIDFM/FMUL, Lisbon, PT, a PAD study where the capacity of UCX® to induce neo-vascularization and improve blood flow was evaluated. The angiogenic response to the administration of UCX® cells in a mouse model of hind limb ischemia was evaluated by analysing perfusion recovery, capillary density and collateral vessel density. Ischemic mice that were administered with UCX® presented a significantly higher perfusion recovery than animals in the control group. At 90 days post-ischemia, mice were sacrificed and capillary density analysed. As expected, ischemia induced capillary density in all experimental groups. However, the results clearly showed that the capillary density in ischemic muscles administered with UCX® was higher when compared to ischemic muscles from untreated mice. According to ECBio’s data, the mice treated with UCX® a dramatic CVD increase in response to ischemia when compared to the untreated mice. These outstanding results led ECBio to further analyse the molecular mechanisms by which UCX® induces angiogenesis, mice were treated with UCX® after ischemia induction. At day 70 post ischemia, mice were sacrificed and gastrocnemius muscles were collected. The mRNA expression of several pro-angiogenic factors and their receptors were assessed by qRT- PCR in ECs isolated from gastrocnemius muscle by Laser Capture Microscope. Our results show that the expression of two specific pro-angiogenic markers is up-regulated in ECs removed from ischemic muscles where UCX® were locally administered, in contrast to ECs isolated from control ischemic muscles. Taken together, the work shows, for the first time, that UCX® in the setting of experimentally induced hind limb ischemia could stimulate angiogenesis and arteriogenesis and thereby improve blood perfusion in the ischemic limb. These results suggest that UCX® could have clinical use in the treatment of lower limb vascular insufficiency. The results have been submitted for publication and have settled the grounds for ECBio’s PAD clinical programme.

In turn, upon myocardial infarction (MI), a cascade of biochemical and morphologic events known as cardiac remodeling takes place in the heart, leading to the formation of a nonfunctional scar in place of the damaged myocardium. Cell therapies have been investigated to direct cardiac response towards efficient repair and/or myocardial functional improvement. In this sense, ECBio and INEBs previous work showed that UCX® is able to preserve cardiac function and attenuate cardiac remodelling after intramyocardial transplantation in a MI murine model through paracrine mechanisms ( Envisioning end user safety, a protocol adaptation is being established in order to produce UCX® as an advanced therapy medicinal product (ATMP – Herein we evaluate the long-term therapeutic effect of acutely injected UCX®-ATMP as a cell therapy to attenuate the aftermath of myocardial infarction on chronic murine (INEB, Univ. of Porto) and acute porcine (Hosp. São João, Univ. of Porto) models for myocardial infarction.

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Delivery of UCX® into the peri-infarction region attenuated cardiac remodeling following MI. (A) Masson’s Trichrome stain on histological sections representative of the LV highlight the collagen deposition (blue) on the infarcted region. Thinning of the LV wall and LV dilatation was observed in the UCX® as well as in the vehicle experimental groups. (B) MI size was quantified by two different methods: the area measurement and the midline length measurement and no significant differences were identified between groups. When compared to non-manipulated animals, the UCX®-transplanted group shows a smaller reduction on of the LV wall thickness (C) and a less pronounced LV dilation (D) than the vehicle group, indicative of UCX®-mediated tapering of cardiac remodeling following MI. *P<0.05. Scale bar = 2mm.