Identifying Key Drivers in the Pathogenesis of Martorell Hypertensive Ischaemic Leg Ulcer: A Comparative Analysis with Chronic Venous Leg Ulcer.

Martorell hypertensive ischaemic leg ulcer (Martorell HYTILU) is a rare but significant cause of distal leg ulcers. Although hypertension and diabetes are known factors in its development, the precise pathogenesis of Martorell HYTILU remains elusive. To reach a better understanding of Martorell HYTILU, transcriptomic analysis was conducted through RNA sequencing and immunohistochemical comparison of Martorell HYTILU (n = 17) with chronic venous ulcers (n = 4) and healthy skin (n = 4). Gene expression analysis showed a marked activation of immune-related pathways in both Martorell HYTILU and chronic venous ulcers compared with healthy skin. Notably, neutrophil activity was substantially higher in Martorell HYTILU. While pathway analysis revealed a mild downregulation of several immune pathways in Martorell HYTILU compared with chronic venous ulcers, keratinization, cornification, and epidermis development were significantly upregulated in Martorell HYTILU. Additionally, STAC2, a gene encoding for a protein promoting the expression of the calcium channel Cav1.1, was significantly upregulated in Martorell HYTILU and was detected perivascularly in situ (Martorell HYTILU n = 24; chronic venous ulcers n = 9, healthy skin n = 11). The high expression of STAC2 in Martorell HYTILU suggests that increased calcium influx plays an important role in the pathogenesis of the disease. Consequently, calcium channel antagonists could be a promising treatment avenue for Martorell HYTILU.

Phosphorylated cingulin localises GEF-H1 at tight junctions to protect vascular barriers in blood endothelial cells.

Dysfunction of vascular barriers is a critical step in inflammatory diseases. Endothelial tight junctions (TJs) control barrier function, and the cytoplasmic adaptor protein cingulin connects TJs to signalling pathways. However, local events at TJs during inflammation are largely unknown. In this study, we investigate the local response of TJ adaptor protein cingulin and its interaction with Rho guanine nucleotide exchange factor H1 (GEF-H1, also known as ARHGEF2) upon vascular barrier disruption to find a new approach to counteract vascular leak. Based on transendothelial-electrical-resistance (TEER) measurements, cingulin strengthened barrier integrity upon stimulation with histamine, thrombin and VEGF. Cingulin also attenuated myosin light chain 2 (MLC2; also known as MYL2) phosphorylation by localising GEF-H1 to cell junctions. By using cingulin phosphomutants, we verified that the phosphorylation of the cingulin head domain is required for its protective effect. Increased colocalisation of GEF-H1 and cingulin was observed in the vessels of vasculitis patients compared to those in healthy skin. Our findings demonstrate that cingulin can counteract vascular leak at TJs, suggesting the existence of a novel mechanism in blood endothelial cells that protects barrier function during disease.

Development of a localization-based algorithm for the prediction of leg ulcer etiology.

BACKGROUND: Diagnostic work-up of leg ulcers is time- and cost-intensive. This study aimed at evaluating ulcer location as a diagnostic criterium and providing a diagnostic algorithm to facilitate differential diagnosis. PATIENTS AND METHODS: The study consisted of 277 patients with lower leg ulcers. The following five groups were defined: Venous leg ulcer, arterial ulcers, mixed ulcer, arteriolosclerosis, and vasculitis. Using computational surface rendering, predilection sites of different ulcer types were evaluated. The results were integrated in a multinomial logistic regression model to calculate the likelihood of a specific diagnosis depending on location, age, bilateral involvement, and ulcer count. Additionally, neural network image analysis was performed. RESULTS: The majority of venous ulcers extended to the medial malleolar region. Arterial ulcers were most frequently located on the dorsal aspect of the forefoot. Arteriolosclerotic ulcers were distinctly localized at the middle third of the lower leg. Vasculitic ulcers appeared to be randomly distributed and were markedly smaller, multilocular and bilateral. The multinomial logistic regression model showed an overall satisfactory performance with an estimated accuracy of 0.68 on unseen data. CONCLUSIONS: The presented algorithm based on ulcer location may serve as a basic tool to narrow down potential diagnoses and guide further diagnostic work-up.

Molecular mechanism of amyloidogenic mutations in hypervariable regions of antibody light chains.

Systemic light chain (AL) amyloidosis is a fatal protein misfolding disease in which excessive secretion, misfolding, and subsequent aggregation of free antibody light chains eventually lead to deposition of amyloid plaques in various organs. Patient-specific mutations in the antibody VL domain are closely linked to the disease, but the molecular mechanisms by which certain mutations induce misfolding and amyloid aggregation of antibody domains are still poorly understood. Here, we compare a patient VL domain with its nonamyloidogenic germline counterpart and show that, out of the five mutations present, two of them strongly destabilize the protein and induce amyloid fibril formation. Surprisingly, the decisive, disease-causing mutations are located in the highly variable complementarity determining regions (CDRs) but exhibit a strong impact on the dynamics of conserved core regions of the patient VL domain. This effect seems to be based on a deviation from the canonical CDR structures of CDR2 and CDR3 induced by the substitutions. The amyloid-driving mutations are not necessarily involved in propagating fibril formation by providing specific side chain interactions within the fibril structure. Rather, they destabilize the VL domain in a specific way, increasing the dynamics of framework regions, which can then change their conformation to form the fibril core. These findings reveal unexpected influences of CDR-framework interactions on antibody architecture, stability, and amyloid propensity.

Topically administered purified clinoptilolite-tuff for the treatment of cutaneous wounds: A prospective, randomised phase I clinical trial.

In an ageing society, chronic ulcers pose an increasingly relevant healthcare issue associated with significant morbidity and an increasing financial burden. Hence, there is an unmet medical need for novel, cost-effective therapies that improve healing of chronic cutaneous wounds. This prospective, randomised, open-label, phase I trial investigated the safety and tolerability of topically administered purified clinoptilolite-tuff (PCT), mainly consisting of the naturally occurring zeolite-mineral clinoptilolite, in artificial wounds in healthy male volunteers compared to the standard of care (SoC). We found that topically administered PCT was safe for therapeutic application in acute wounds in healthy male volunteers. No significant differences in wound healing or wound conditions were observed compared to SoC-treated wounds. However, we found a significantly higher proportion of CD68-positive cells and a significantly lower proportion of α-smooth muscle actin-positive cells in PCT-treated wounds. Scanning electron microscopy revealed PCT particles in the restored dermis in some cases. However, these did not impede wound healing or clinical symptoms. Hence, purified PCT could represent an attractive, cost-effective wound treatment promoting the process of healing.

Comparative analysis of endovenous laser ablation versus ultrasound-guided foam sclerotherapy for the treatment of venous leg ulcers.

Endovenous thermal and non-thermal therapeutic approaches have become standard of care for the treatment of venous insufficiency. However, comparative studies on its use in the population of venous leg ulcer patients are scarce. The present study aimed at a comparison of the efficacy of endovenous laser ablation (EVLA) and ultrasound-guided foam sclerotherapy (UGFS) for the treatment of venous leg ulcers (VUs). We retrospectively analyzed patient records of 68 patients with active VUs (C6 of the CEAP-classification), who underwent EVLA (n = 33) or UGFS (n = 35) between January 2001 and January 2021. In 68 patients, 97 venous segments (GSV: 43, SSV: 17, NSV: 37) were treated. Ulcer surface area at initial presentation did not differ significantly between both treatment groups (EVLA: 7.7 ± 10.7 vs. UGFS: 8.5 ± 16.3 cm2 ; p = 0.73). No significant difference regarding patient characteristics was found, with the exception of age, as patients receiving UGFS treatment were significantly older (EVLA: 61 ± 17 vs. UGFS: 70 ± 14 years; p = 0.018). The rate of ulcer resolution was not significantly different between EVLA and UGFS groups (97.0% vs. 85.7%; p = 0.20). Also, the mean time to complete ulcer healing after endovenous intervention was comparable (EVLA: 59 ± 37 vs. UGFS: 63 ± 41 days; p = 0.68). However, the relapse rate was significantly higher for UGFS than for EVLA treated patients (31.4% vs. 3.0%; p = 0.002). Taken together, rates of ulcer resolution and ulcer healing time after endovenous intervention were comparable between both treatment modalities. Nevertheless, a significantly higher relapse rate was observed in UGFS treated patients.

Effectiveness of narrowband UVB phototherapy and psoralen plus UVA photochemotherapy in the treatment of generalized lichen planus: Results from a large retrospective analysis and an update of the literature.

BACKGROUND: The aim of this retrospective study was to compare the efficacy and safety of different phototherapeutic modalities in the treatment of cutaneous lichen planus (LP). METHODS: We retrospectively analyzed the chart data of 53 patients with generalized LP who had been subjected to narrowband UVB (NB-UVB) or photochemotherapy (PUVA) between January 1997 and April 2020. Of these, 30 patients had received NB-UVB, 18 patients oral PUVA and 5 patients bath PUVA. RESULTS: Fifty patients completed a full treatment course. The percentage of patients with a complete (>90% clearing) or good (51%-90% clearing) response was similar for NB-UVB versus PUVA (86.2% vs. 90.5%; P = 1.00). The number of exposures required for obtaining a complete or good response was also comparable for both treatment groups (NB-UVB: 28.9 ± 12.3 vs. PUVA: 25.4 ± 10.1; P = .209). Adverse events, in particular gastrointestinal upsets, were recorded in 26.1% of patients treated with oral PUVA while none were observed with NB-UVB. CONCLUSION: The therapeutic outcome and the number of treatments required for achieving a complete or good response were comparable for NB-UVB and PUVA; however, PUVA therapy was associated with a substantially higher rate of moderate adverse events.

Differentiating Arteriolosclerotic Ulcers of Martorell from Other Types of Leg Ulcers Based on Vascular Histomorphology.

Clinical differential diagnosis of arteriolosclerotic ulcers of Martorell is challenging due to the lack of clearly affirmative instrument-based diagnostic criteria. The aim of this study was to develop vascular histomorphological diagnostic criteria differentiating Martorell ulcers from other types of leg ulcers. The histomorphology of patients diagnosed with arteriolosclerotic ulcers of Martorell (n = 67) was compared with that of patients with venous leg ulcers, necrotizing leukocytoclastic vasculitis, pyoderma gangrenosum, and non-ulcerative controls (n = 15 each). In a multivariable logistic regression model, the rates of arteriolar calcification (odds ratio (OR) 42.71, 95% confidence interval (CI) 7.43-443.96, p < 0.001) and subendothelial hyalinosis (OR 29.28, 95% CI 4.88-278.21, p <0.001) were significantly higher in arteriolosclerotic ulcers of Martorell. Arteriolar cellularity was significantly lower in Martorell ulcers than in controls (OR 0.003, 95 CI < 0.001-0.97, p = 0.05). However, the wall-to-lumen ratio was similar in all ulcers (OR 0.975, 95% CI 0.598-2.04, p =0.929). Based on the Youden index, a wall cellularity of < 0.24 cells/100 µm2 was determined as the optimum cut-off point (sensitivity 0.955, specificity 0.944). Thus, arteriolar calcification, subendothelial hyalinosis, and arteriolar cellularity revealed high discriminatory power for arteriolosclerotic ulcers of Martorell.

Drug-induced photosensitivity: culprit drugs, potential mechanisms and clinical consequences.

Drug-induced photosensitivity, the development of phototoxic or photoallergic reactions due to pharmaceuticals and subsequent exposure to ultraviolet or visible light, is an adverse effect of growing interest. This is illustrated by the broad spectrum of recent investigations on the topic, ranging from molecular mechanisms and culprit drugs through epidemiological as well as public health related issues to long-term photoaging and potential photocarcinogenic consequences. The present review summarizes the current state of knowledge on the topic while focusing on culprit drugs and long-term effects. In total, 393 different drugs or drug compounds are reported to have a photosensitizing potential, although the level of evidence regarding their ability to induce photosensitive reactions varies markedly among these agents. The pharmaceuticals of interest belong to a wide variety of drug classes. The epidemiological risk associated with the use of photosensitizers is difficult to assess due to under-reporting and geographical differences. However, the widespread use of photosensitizing drugs combined with the potential photocarcinogenic effects reported for several agents has major implications for health and safety and suggests a need for further research on the long-term effects.

Peptides in proteins.

During evolution C-terminal peptide extensions were added to proteins on the gene level. These convey additional functions such as interaction with partner proteins or oligomerisation. IgM antibodies and molecular chaperones are two prominent examples discussed.

A peptide extension dictates IgM assembly.

Professional secretory cells can produce large amounts of high-quality complex molecules, including IgM antibodies. Owing to their multivalency, polymeric IgM antibodies provide an efficient first-line of defense against pathogens. To decipher the mechanisms of IgM assembly, we investigated its biosynthesis in living cells and faithfully reconstituted the underlying processes in vitro. We find that a conserved peptide extension at the C-terminal end of the IgM heavy (Ig-µ) chains, termed the tailpiece, is necessary and sufficient to establish the correct geometry. Alanine scanning revealed that hydrophobic amino acids in the first half of the tailpiece contain essential information for generating the correct topology. Assembly is triggered by the formation of a disulfide bond linking two tailpieces. This induces conformational changes in the tailpiece and the adjacent domain, which drive further polymerization. Thus, the biogenesis of large and topologically challenging IgM complexes is dictated by a local conformational switch in a peptide extension.

A single residue switch reveals principles of antibody domain integrity.

Despite their importance for antibody architecture and design, the principles governing antibody domain stability are still not understood in sufficient detail. Here, to address this question, we chose a domain from the invariant part of IgG, the CH2 domain. We found that compared with other Ig domains, the isolated CH2 domain is a surprisingly unstable monomer, exhibiting a melting temperature of ∼44 °C. We further show that the presence of an additional C-terminal lysine in a CH2 variant substantially increases the melting temperature by ∼14 °C relative to CH2 WT. To explore the molecular mechanism of this effect, we employed biophysical approaches to probe structural features of CH2. The results revealed that Lys101 is key for the formation of three secondary structure elements: the very C-terminal ß-strand and two adjacent α-helices. We also noted that a dipole interaction between Lys101 and the nearby α-helix, is important for stabilizing the CH2 architecture by protecting the hydrophobic core. Interestingly, this interaction between the α-helix and C-terminal charged residues is highly conserved in antibody domains, suggesting that it represents a general mechanism for maintaining their integrity. We conclude that the observed interactions involving terminal residues have practical applications for defining domain boundaries in the development of antibody therapeutics and diagnostics.

Efficacy of In Vivo Electroporation-Mediated IL-10 Gene Delivery on Survival of Skin Flaps.

Despite advances in understanding the underlying mechanisms of flap necrosis and improvement in surgical techniques, skin flap necrosis after reconstructive surgery remains a crucial issue. We investigated the efficacy of electroporation-mediated IL-10 gene transfer to random skin flap with an aim to accelerate wound healing and improve skin flap survival. Nine male Wistar rats (300-330 g) were divided in two groups (a) control group (n = 5), only surgery no gene transfer, and (b) experimental group, received electroporation-mediated IL-10 gene transfer 24 h before the surgery as prophylaxis (n = 4). Random skin flap (McFarlane) was performed in both groups. Planimetry, Laser Doppler imaging, and immunohistochemistry were used to evaluate the effect of IL-10 gene transfer between study groups at day 7. Electroporation-mediated IL-10 gene transfer decreased percentage of flap necrosis (p value = 0.0159) and increased cutaneous perfusion compared to the control group (p value = 0.0159). In addition, Spearman's rank correlation showed a significant negative correlation between percentage of flap necrosis and Laser Index (p value = 0.0083, r -0.83, respectively). Furthermore, significantly higher mean CD31+ vessel density was detected in the experimental group compared to the control group (p value = 0.0159). Additionally, semi-quantitative image analysis showed lower inflammatory cell count in experimental group compared to control group (p value = 0.0317). In vivo electroporation-mediated IL-10 gene transfer reduced necrosis, enhanced survival and vascularity in the ischemic skin flap.

Gestational Age-Dependent Fetal Fluid Dynamics in the Ovine Developmental Model: Establishment of Surrogate Markers for the Differentiation of Stem Cell Origin.

The ovine developmental model represents the standard in vivo model for studies involving maternofetal physiology, amniotic fluid (AF) research, and fetal cell therapy prior to human clinical use. Although being close to the human fetal anatomy, 2 separate extraembryonic fluid compartments remain during gestation, known as the amnion and the allantois. A clear distinction between AF versus allantoic fluid (AL) is therefore indispensable for correct scientific conclusions with regard to human translation. In the presented study, the biochemical composition of AF and AL was evaluated in ovine gravid uteri postmortem (n = 31) over the entire gestation. Four parameters, consisting of Na+, Cl-, Mg2+, and total protein, have been found to allow for specific discrimination of the 2 fetal fluids at all gestational phases and therefore as potential surrogate parameters for gestational age. In addition, volumetric changes of the developing fetus and the 2 fetal fluid cavities were analyzed by contrast-enhanced computed tomography (n = 12). AF showed a significant, linear volumetric increase over gestation, whereas AL volume maintained relatively static independent of gestational age. These results serve as a basis for future studies by providing surrogate markers enabling a reliable distinction of isolated fetal fluids and contained cells in the ovine developmental model over the entire gestation.

25-Hydroxyvitamin-D3 serum modulation after use of sunbeds compliant with European Union standards: A randomized open observational controlled trial.

BACKGROUND: Regular use of sunbed exposure has been reported to increase 25-hydroxyvitamin-D3 [25(OH)D] serum levels. However, the influence of sunbeds compliant with the recent European Union standard EN-60335-2-27 on 25(OH)D serum levels is unknown. OBJECTIVE: We investigated the impact of standard sunbed use compliant with the European Union standard on 25(OH)D serum modulation and well-being. METHODS: In a randomized controlled study, 25(OH)D serum levels were measured at enrollment, after 1 week, and after completion of the 12-week period of sunbed use with twice weekly exposure and compared with the control group without any sunbed exposure. RESULTS: In the sunbed intervention group (N = 31), a 27% increase of mean 25(OH)D levels was noted 1 week after starting sunbed use (P < .01). However, after 12 weeks, mean 25(OH)D levels had declined and were no longer different from baseline (P = .06). After 12 weeks, 25(OH)D levels did not differ between the intervention and control group (P = .36). Also the 5-item World Health Organization Well-Being Index score did not differ between the sunbed and control groups (P = .19). LIMITATIONS: For ethical reasons recruitment was limited to persons actively seeking sunbed exposure. CONCLUSIONS: Standard use of sunbeds compliant with the European Union standard induced a transient increase of 25(OH)D levels, whereas no change in well-being was observed.

Interventional Closure of Secundum Type Atrial Septal Defects in Infants Less Than 10 Kilograms: Indications and Procedural Outcome.

AIMS: This study set out to assess indications, feasibility, complications, and clinical outcome of percutaneous transcatheter device closure of atrial septal defects (ASDs) in infants with a bodyweight below 10 kg. METHODS AND RESULTS: Retrospective single center chart and echocardiography review study from 8/2005-12/2013. Twenty-eight children with ASD (13 female) with a median age of 1.15 years (0.2-2.8) and a median weight of 7.2 kg (4.5-9.9) were analyzed. Indications for early ASD closure were failure to thrive (n = 15, 54%), bronchopulmonary dysplasia (BPD) with supplemental oxygen dependency (n = 7, 25%), and genetic syndromes with suspected pulmonary hypertension (n = 12, 43%). Device implantation was successful in all patients without any periprocedural mortality or major complication. Clinical outcome after a median follow-up period of 2.1 years (0.25-7.3) revealed no residual shunt and a significant decrease of right ventricular volume load. Patients with pulmonary hypertension experienced a significant reduction of pulmonary artery/RV pressure. Patients also showed decreased supplemental oxygen dependency and less cardiac medications, but no significant "catch-up growth" in those with failure to thrive. CONCLUSION: Interventional ASD closure in children weighing less than 10 kg can be performed without any additional major risks and shows a favorable outcome, especially in selected patients with significant non cardiac co-morbidities.

PGA (polyglycolic acid)-P4HB (poly-4-hydroxybutyrate)-Based Bioengineered Valves in the Rat Aortic Circulation.

BACKGROUND AND AIM OF THE STUDY: Bioengineered living autologous valves with remodeling and growth capacity represent a promising concept for future cardiac and venous valve repair. A meticulous understanding of the mechanisms involved in recellularization and remodeling is essential for the safe and efficient clinical translation of this technology. In this context, the first investigations of bioengineered vascular grafts in immune-incompetent or transgenic rodents represented an important step. However, the in-vivo assessment of bioengineered synthetic scaffold-based (biodegradable) valve replacements in rodent models has not been achieved to date. METHODS: Miniaturized monocuspid PGA (polyglycolic acid)-P4HB (poly-4-hydroxybutyrate)-based valves were created, incorporated into metallic stents (length 2.0 mm, diameter 1.1 mm) and introduced into catheter-based implantation devices. Wistar outbred rats (n = 8) underwent a laparotomy, abdominal aorta arteriotomy and valve delivery into the abdominal aorta. Valve placement and function were evaluated following deployment using ultrasound (Doppler- and M-mode). Explanted tissues were analyzed both macroscopically and histopathologically. RESULTS: No significant physiological or hemodynamic changes were observed, including heart rate, pressure gradients, velocity values and cardiac output before and after valve implantation. The cross-sectional area at the level of the stented valve was reduced by 22%. Valvular leaflet oscillation was observed in two animals, and thrombus formation in the stent was observed in one animal. Histological evaluation revealed cellular infiltration within 3 h in vivo, and no signs of thrombus deposition on the valvular surface. CONCLUSIONS: This study demonstrated the technical feasibility of the transcatheter implantation of bioengineered stented miniaturized valves into the infrarenal rat aorta, without affecting the animal's physiological and hemodynamic variables and with valvular oscillation in part of the implants. These results could serve as a basis for the implementation of a chronic rat in-vivo model for mechanistic studies in bioengineered valvular tissues under systemic hemodynamic conditions. Video 1: 2D ultrasonographic projection revealing graft's leaflet oscillation.

Hemodynamic Assessment of a Murine Heterotopic Biventricularly Loaded Cardiac Transplant in vivo Model.

BACKGROUND: Heterotopic heart transplantation (HHT) in rodent animal models represents an important technique enabling studies on organ transplantation immunology and pharmaceutical development. Recent investigations used nonworking HHT designs, with the left ventricle (LV) bypassed in the anastomosis system. In spite of their principal success, the lack of orthogonal ventricular filling leads to myocardial atrophy. However, when focusing on the cellular and molecular mechanisms involved in the in vivo remodeling of the myocardium or cell-based cardiovascular implants, a nonworking model is suboptimal as it lacks the native-analogous hemodynamic and metabolic situation. Here we present the hemodynamic and electrical assessment of a biventricularly loaded murine HHT method without the need for a combined heart-lung transplantation approach. METHODS: Heterotopic transplantations (n = 13) were performed on C57BL/6J-(H-2b) inbred mice (n = 13 donors, n = 13 recipients) by creating end-to-side anastomoses between the donors' cranial vena cava (CrVC) and the recipients' abdominal caudal vena cava (CVC), between the donors' ascending aorta and the recipients' abdominal aorta (aAo), and between the grafts' pulmonary trunk and the left atrium. After transplantation, a hemodynamic assessment using echocardiography (including 2D speckle tracking analysis) and electrocardiography was performed. RESULTS: The loaded HHT procedure in the mice was performed with an overall success rate of 61%. In 3 of the remaining 5 cases, only atrial function was restored. The median duration of the entire surgical procedure for the recipient animal was 190 (IQR 180-250) min. The mean heart rate in the loaded HHT group was 355 ± 6 bpm in comparison to the control group with an in situ heart rate of 418 ± 61 bpm. A native-like closing and opening pattern of the aortic and mitral valves (visible on both 2D and M-mode images) was observed, confirming a native-analogous loading of the LV. Pulsed-wave Doppler provided visualization of the flow across the region of anastomoses between the pulmonary trunk and the left atrium, reaching a mean maximum velocity of 382 ± 12 mm/s. Exemplary 2D speckle tracking analysis of the LV free wall and interventricular septum revealed some differences in vector directions in one animal when compared to the orthotopic native heart, indicating an asynchronous movement of the LV. CONCLUSIONS: These results demonstrate the technical (micro)surgical feasibility of a fully loaded HHT procedure in the murine model without using a combined heart-lung transplantation approach. The acute hemodynamic performance of the HHT grafts approximated the native orthotopic situation. This model may open up new options for the investigation of cellular and molecular questions in the murine cardiovascular in vivo system in the near future.

Cardiovascular Regenerative Technologies: Update and Future Outlook.

In the effort of improving treatment for cardiovascular disease (CVD), scientists struggle with the lack of the regenerative capacities of finally differentiated cardiovascular tissues. In this context, the advancements in regenerative medicine contributed to the development of cell-based therapies as well as macro- and micro-scale tissue-engineering technologies. The current experimental approaches focus on different regenerative strategies including a broad spectrum of techniques such as paracrine-based stimulation of autologous cardiac stem cells, mesenchymal cell injections, 3D microtissue culture techniques and vascular tissue-engineering methods. These potential next-generation strategies are leading the way to a revolution in addressing CVD, and numerous studies are now undertaken to assess their therapeutic value. With this review, we provide an update on the current research directions, on their major challenges, limitations, and achievements.