Functionalised peptide hydrogel for the delivery of cardiac progenitor cells.

Heart failure (HF) remains one of the leading causes of death worldwide; most commonly developing after myocardial infarction (MI). Since adult cardiomyocytes characteristically do not proliferate, cells lost during MI are not replaced. As a result, the heart has a limited regenerative capacity. There is, therefore, a need to develop novel cell-based therapies to promote the regeneration of the heart after MI. The delivery and retention of cells at the injury site remains a significant challenge. In this context, we explored the potential of using an injectable, RGDSP-functionalised self-assembling peptide - FEFEFKFK - hydrogel as scaffold for the delivery and retention of rat cardiac progenitor cells (CPCs) into the heart. Our results show that culturing CPCs in vitro within the hydrogel for one-week promoted their spontaneous differentiation towards adult cardiac phenotypes. Injection of the hydrogel on its own, or loaded with CPCs, into the rat after injury resulted in a significant reduction in myocardial damage and left ventricular dilation.

Enhancement of peri-implant bone osteogenic activity induced by a peptidomimetic functionalization of titanium.

Osteoblast cell adhesion to the extracellular matrix is established through two main pathways: one is mediated by the binding between integrin and a minimal adhesion sequence (RGD) on the extracellular protein, the other is based on the interactions between transmembrane proteoglycans and heparin-binding sequences found in many matrix proteins. The aim of this study is the evaluation in an in vivo endosseous implant model of the early osteogenic response of the peri-implant bone to a biomimetic titanium surface functionalized with the retro-inverso 2DHVP peptide, an analogue of Vitronectin heparin binding site. The experimental plan is based on a bilateral study design of Control and 2DHVP implants inserted respectively in the right and left femur distal metaphysis of adult male Wistar rats (n=16) weighing about 300grams and evaluated after 15days. Fluorochromic bone vital markers were given in a specific time frame, in order to monitor the dynamic of new bone deposition. The effect inducted by the peptidomimetic coating on the surrounding bone were qualitatively and quantitatively evaluated by means of static and dynamic histomorphometric analyses performed within three concentric and subsequent circular Regions of Interest (ROI) of equivalent thickness (220µm), ROI1 adjacent to the interface, ROI2, the middle, and ROI3 the farthest. The data indicated that these functionalized implants stimulated a higher bone apposition rate (p<0,01) and larger and rapid osteoblast activation in terms of mineralizing surface within ROI1 compared to the control (p<0,01). These higher osteoblast recruitment and activation leads to a greater bone-to-implant contact reached for DHVP samples (p<0,5). This represents an initial stimulus of the osteogenic activity that might results in a faster and better osteointegration process.

Stanozolol-soaked grafts enhance new bone formation in rat calvarial critical-size defects.

Androgen hormones play a significant role in regulating bone morphogenesis and in maintaining bone homeostasis throughout life. This study aimed to investigate the local effects of the non-aromatizable androgen stanozolol (ST) on bone regeneration in rats. Bilateral critical-size defects were created in the parietal bone of 26 male Wistar rats: the defect on one side was filled with a deproteinized bovine bone scaffold (DBB) soaked in ST solution (test) and the contralateral with DBB alone (control). Samples were collected at one month and three months. Histomorphometry revealed a significantly higher new bone formation (NB) (24.41% ± 4.14% versus 15.01% ± 2.43%, p < 0.05) and mineral apposition rate (MAR) (9.20 µm/day ± 0.37 versus 6.50 µm/day ± 1.09, p < 0.05) in the test versus control group at one month. Accordingly, real time-polymerase chain reaction revealed a consistently higher Runx2 expression in test samples (fold change test/control: 4.50 ± 1.17, p ≤ 0.05). No morphometrical differences between groups were detected at three months (p > 0.05). However, test samples were characterized by an increase in blood capillary density from one month (11.43 n mm-2 ± 2.01) to three months (28.26 n mm-2 ± 5.62), providing evidence of a vital remodeling tissue. Control samples presented a decrease of anti-Osterix (SP7)/anti-osteocalcin (BGLAP) (3.9 n mm-2 ± 0.32 versus 1.01 n mm-2 ± 0.20) and alkaline phosphatase (ALP) (12.14 n mm-2 ± 6.29 versus 6.29 n mm-2 ± 2.73) immunohistochemical-positive elements, which was suggestive of a stabilized healing phase. Based on these observations, local ST administration boosted bone regeneration in rat calvarial critical-size defects at one month. This study showed the potential of local steroid delivery in bone regeneration.

Social defeat and isolation induce clear signs of a depression-like state, but modest cardiac alterations in wild-type rats.

Adverse social environments play a relevant role in the onset and progression of mood disorders. On the other hand, depression is an independent risk factor for cardiovascular morbidity. This study was aimed at (i) corroborating the validity of a rat model of depression based on a negative social episode followed by social isolation and (ii) verifying its impact on cardiac function and structure. Pair housed, wild-type Groningen rats (Rattus norvegicus) were implanted with radiotransmitters for ECG, temperature and activity recordings. They were either exposed to a social defeat episode followed by 4-week isolation or left undisturbed with their female partners. The social challenge induced a series of biological changes that are commonly taken as markers of depression in rats, including decreased body weight gain and reduced preference for sucrose consumption, functional and structural changes of the hypothalamic-pituitary-adrenocortical axis, increased anxiety in the elevated plus maze test. The cardiovascular alterations consisted in (i) transitory heart rate circadian rhythm alterations, (ii) lack of habituation of cardiac autonomic responsivity (tachycardia and vagal withdrawal) to an acute stressor, and (iii) moderate hypertrophy affecting the right ventricle of the heart. These results indicate that a depression-like state induced via this model of social challenge was associated with a few modest cardiovascular changes. Further studies are required to confirm the validity of this rat model of depression as a valid preclinical approach to the comprehension of the biological substrates underlying depression-cardiovascular comorbidity.

Early maternal separation has mild effects on cardiac autonomic balance and heart structure in adult male rats.

Early life adverse experiences have long-term physiologic and behavioral effects and enhance stress sensitivity. This study examined the effects of maternal separation (MS) on cardiac stress responsivity and structure in adulthood. Male Wistar rats were separated from the dams for 3 h per day from postnatal days 2 through 15. When exposed to 5-day intermittent restraint stress (IRS) as adults, MS, and control rats showed similar acute modifications of cardiac sympathovagal balance, quantified via heart rate variability analysis. In addition, MS had no effect on cardiac pacemaker intrinsic activity (as revealed by autonomic blockade with scopolamine and atenolol) and did not affect the circadian rhythmicity of heart rate, neither before nor after IRS. However, MS differed from control rats in cardiac parasympathetic drive following IRS, which was heightened in the latter but remained unchanged in the former, both during the light and dark phases of the daily rhythm. The evaluation of adult cardiac structure indicated that stress experienced during a crucial developmental period induced only modest changes, involving cardiomyocyte hypertrophy, increased density of vascular structures, and myocardial fibrosis. The mildness of these functional-structural effects questions the validity of MS as a model for early stress-induced cardiac disease in humans.

Resident cardiac stem cells.

The introduction of stem cells in cardiology provides new tools in understanding the regenerative processes of the normal and pathologic heart and opens new options for the treatment of cardiovascular diseases. The feasibility of adult bone marrow autologous and allogenic cell therapy of ischemic cardiomyopathies has been demonstrated in humans. However, many unresolved questions remain to link experimental with clinical observations. The demonstration that the heart is a self-renewing organ and that its cell turnover is regulated by myocardial progenitor cells offers novel pathogenetic mechanisms underlying cardiac diseases and raises the possibility to regenerate the damaged heart. Indeed, cardiac stem progenitor cells (CSPCs) have recently been isolated from the human heart by several laboratories although differences in methodology and phenotypic profile have been described. The present review points to the potential role of CSPCs in the onset and development of congestive heart failure and its reversal by regenerative approaches aimed at the preservation and expansion of the resident pool of progenitors.

Identification of the prosurvival activity of nerve growth factor on cardiac myocytes.

Neurotrophins (NTs) control neuron survival and regeneration. Recent research showed that NTs possess cardiovascular actions. In this study, we investigated the hypothesis that the NT nerve growth factor (NGF) prevents cardiomyocyte apoptosis. We demonstrated that cultured rat neonatal cardiomyocytes (RNCMs) produce NGF and express its trkA (tropomyosin-related receptor A (NGF high-affinity receptor)) receptor. RNCMs given a neutralizing antibody for NGF or the trkA inhibitor K252a underwent apoptosis, thus suggesting that NGF is an endogenous prosurvival factor for cardiomyocytes. Adenovirus (Ad)-mediated NGF overexpression protected RNCMs from apoptosis induced by either hypoxia/reoxygenation or angiotensin II (AngII). Similarly, recombinant NGF inhibited AngII-induced apoptosis in isolated rat adult cardiomyocytes. Finally, in a rat model of myocardial infarction, NGF gene transfer promoted cardiomyocyte survival. In RNCMs, recombinant NGF induced trkA phosphorylation, followed by Ser473 phosphorylation and nuclear translocation of phospho-protein kinase B (Akt). In response to Akt activation, Forkhead transcription factors Foxo-3a and Foxo-1 were phosphorylated and excluded from the nucleus. The prosurvival effect of adenoviral vector carrying the human NGF gene was inhibited in vitro by K252a, LY294002 (a pan-phosphatidyl inositol 3-kinase - PI3K - inhibitor), an Akt small interfering RNA, and adenoviruses carrying a dominant negative mutant form of Akt (Ad.DN.Akt) or an Akt-resistant Foxo-3a (Ad.AAA-Foxo-3a). These results newly demonstrate the cardiac prosurvival action of NGF and provide mechanistic information on the signaling pathway, which encompasses trkA, PI3K-Akt, and Foxo.

Benfotiamine accelerates the healing of ischaemic diabetic limbs in mice through protein kinase B/Akt-mediated potentiation of angiogenesis and inhibition of apoptosis.

AIMS/HYPOTHESIS: Benfotiamine, a vitamin B1 analogue, reportedly prevents diabetic microangiopathy. The aim of this study was to evaluate whether benfotiamine is of benefit in reparative neovascularisation using a type I diabetes model of hindlimb ischaemia. We also investigated the involvement of protein kinase B (PKB)/Akt in the therapeutic effects of benfotiamine. METHODS: Streptozotocin-induced diabetic mice, given oral benfotiamine or vehicle, were subjected to unilateral limb ischaemia. Reparative neovascularisation was analysed by histology. The expression of Nos3 and Casp3 was evaluated by real-time PCR, and the activation state of PKB/Akt was assessed by western blot analysis and immunohistochemistry. The functional importance of PKB/Akt in benfotiamine-induced effects was investigated using a dominant-negative construct. RESULTS: Diabetic muscles showed reduced transketolase activity, which was corrected by benfotiamine. Importantly, benfotiamine prevented ischaemia-induced toe necrosis, improved hindlimb perfusion and oxygenation, and restored endothelium-dependent vasodilation. Histological studies revealed the improvement of reparative neovascularisation and the inhibition of endothelial and skeletal muscle cell apoptosis. In addition, benfotiamine prevented the vascular accumulation of advanced glycation end products and the induction of pro-apoptotic caspase-3, while restoring proper expression of Nos3 and Akt in ischaemic muscles. The benefits of benfotiamine were nullified by dominant-negative PKB/Akt. In vitro, benfotiamine stimulated the proliferation of human EPCs, while inhibiting apoptosis induced by high glucose. In diabetic mice, the number of circulating EPCs was reduced, with the deficit being corrected by benfotiamine. CONCLUSIONS/INTERPRETATION: We have demonstrated, for the first time, that benfotiamine aids the post-ischaemic healing of diabetic animals via PKB/Akt-mediated potentiation of angiogenesis and inhibition of apoptosis. In addition, benfotiamine combats the diabetes-induced deficit in endothelial progenitor cells.

Nerve growth factor supplementation reverses the impairment, induced by Type 1 diabetes, of hindlimb post-ischaemic recovery in mice.

AIMS/HYPOTHESIS: Type 1 diabetes increases the risk of peripheral ischaemia and impairs recovery once ischaemia occurs, probably because the healing process is hampered by diabetes-induced endothelial dysfunction. In normoglycaemic mice subjected to limb ischaemia, blockade of nerve growth factor (NGF) compromises reparative angiogenesis. In the present study, we evaluated if expressional alterations of endogenous NGF system components are associated with diabetes-related impairment in neovascularisation. In addition, we tested whether the correction of NGF liabilities benefits post-ischaemic healing of Type 1 diabetic animals. METHODS: Unilateral hindlimb ischaemia was produced in streptozotocin-induced Type 1 diabetic mice. Purified murine NGF (20 microg daily for 14 days) or PBS were injected into ischaemic adductors. Non-diabetic mice given PBS served as controls. Hindlimb blood flow was analysed sequentially for up to 14 days. At necroscopy, adductors were removed for quantification of microvessel density, endothelial cell apoptosis and NGF receptor expression. NGF content was determined by ELISA three days after ischaemia. In vitro, we tested whether NGF protects endothelial cells from apoptosis induced by high glucose and whether vascular endothelial growth factor-A (VEGF-A) is involved in this beneficial effect. RESULTS: Muscles removed from Type 1 diabetic mice showed reduced NGF content and up-regulation of the NGF p75 receptor. NGF supplementation promoted capillarisation and arteriogenesis, reduced apoptosis, and accelerated blood flow recovery. NGF stimulated VEGF-A production by human endothelial cells incubated in high-glucose medium and conferred resistance against high-glucose-induced apoptosis via a VEGF-A-mediated mechanism. CONCLUSIONS/INTERPRETATION: NGF protects endothelial cells from apoptosis induced by Type 1 diabetes and facilitates reparative neovascularisation. The findings may open up new therapeutic options for the treatment of diabetic complications.

Nerve growth factor promotes reparative angiogenesis and inhibits endothelial apoptosis in cutaneous wounds of Type 1 diabetic mice.

AIMS/HYPOTHESIS: The neurotrophin nerve growth factor (NGF) is pro-angiogenic and facilitates wound repair. The present study was conducted to (i) assess the statement of NGF system components in diabetic wounds and (ii) evaluate whether NGF supplementation could prevent impairment of wound neoangiogenesis by diabetes. METHODS: Skin wounds were produced in the interscapular region of streptozotocin-induced diabetic mice. NGF (1 microg per day in PBS) or vehicle was applied onto the ulcers for 3 days after punching. Non-diabetic mice were used as controls. RESULTS: In wounds of untreated diabetic mice, endogenous levels of immunoreactive NGF were lower than those in wounds of non-diabetic mice ( p<0.01). Immunohistochemical analysis showed down-regulation of tyrosine kinase receptor-A (TrkA) and up-regulation of p75 receptor in granulation tissue microvasculature. Local NFG administration prevented diabetes-induced expressional alterations, enhanced reparative capillarisation ( p<0.01), and accelerated wound closure ( p<0.01). This was associated with a three-fold increase in endothelial cell proliferation ( p<0.01), while apoptosis was reduced by 50% ( p<0.05). Quantitative RT-PCR documented a 5.5-fold increase in the expression of vascular endothelial growth factor-A (VEGF-A) by exogenous NGF in diabetic tissues ( p<0.01). In in vitro preparations of human endothelial cells from derma, NGF increased the release of immunoreactive VEGF-A, and reduced high-glucose-induced apoptosis ( p<0.05), the latter effect being inhibited by a VEGF-A receptor-2 antagonist. CONCLUSIONS/INTERPRETATION: Diabetic ulcers display distinct alterations in reparative angiogenesis and in the expression of NGF and its receptors. NGF supplementation corrects endogenous liabilities, facilitates vascular regeneration, and suppresses endothelial apoptosis seemingly via VEGF-A. Our findings unravel new mechanisms responsible for NGF reparative action.