Three dimensional (bio)printing of blood vessels: from vascularized tissues to functional arteries.

Tissue engineering has emerged as a strategy for producing functional tissues and organs to treat diseases and injuries. Many chronic conditions directly or indirectly affect normal blood vessel functioning, necessary for material exchange and transport through the body and within tissue-engineered constructs. The interest in vascular tissue engineering is due to two reasons: (1) functional grafts can be used to replace diseased blood vessels, and (2) engineering effective vasculature within other engineered tissues enables connection with the host's circulatory system, supporting their survival. Among various practices, (bio)printing has emerged as a powerful tool to engineer biomimetic constructs. This has been made possible with precise control of cell deposition and matrix environment along with the advancements in biomaterials. (Bio)printing has been used for both engineering stand-alone vascular grafts as well as vasculature within engineered tissues for regenerative applications. In this review article, we discuss various conditions associated with blood vessels, the need for artificial blood vessels, the anatomy and physiology of different blood vessels, available 3D (bio)printing techniques to fabricate tissue-engineered vascular grafts and vasculature in scaffolds, and the comparison among the different techniques. We conclude our review with a brief discussion about future opportunities in the area of blood vessel tissue engineering.

Approximating scaffold printability utilizing computational methods.

Bioprinting facilitates the generation of complex, three-dimensional (3D), cell-based constructs for various applications. Although multiple bioprinting technologies have been developed, extrusion-based systems have become the dominant technology due to the diversity of materials (bioinks) that can be utilized, either individually or in combination. However, each bioink has unique material properties and extrusion characteristics that affect bioprinting utility, accuracy, and precision. Here, we have extended our previous work to achieve high precision (i.e. repeatability) and printability across samples by optimizing bioink-specific printing parameters. Specifically, we hypothesized that a fuzzy inference system (FIS) could be used as a computational method to address the imprecision in 3D bioprinting test data and uncover the optimal printing parameters for a specific bioink that result in high accuracy and precision. To test this hypothesis, we have implemented a FIS model consisting of four inputs (bioink concentration, printing flow rate, speed, and temperature) and two outputs to quantify the precision (scaffold bioprinted linewidth variance) and printability. We validate our use of the bioprinting precision index with both standard and normalized printability factors. Finally, we utilize optimized printing parameters to bioprint scaffolds containing up to 30 × 106cells ml-1with high printability and precision. In total, our results indicate that computational methods are a cost-efficient measure to improve the precision and robustness of extrusion 3D bioprinting.

A robust quantitative approach for laser speckle contrast imaging perfusion analysis revealed anomalies in the brain blood flow in offspring mice of preeclampsia.

Microcirculation analysis of the brain cortex is challenging because surface perfusion varies rapidly in small space-time regions and is bone protected. The laser speckle contrast imaging (LSCI) technique allows analyzing in vivo brain vascular perfusion generating a large amount of data that requires sophisticated data analytics, making researchers invest much effort in processing. Our research question was whether the reduced placental perfusion model (RUPP) of preeclampsia (PE) was associated with impaired blood perfusion in the offspring's brains. We aimed to develop a robust numerical approach that mainly consisted of applying a signal-processing tool for calculating optimal segmentation and piece-wise fits of the offspring's brain perfusion signals obtained from the LSCI technique. We combined this tool with the usual statistical analysis, implementing both in Matlab software. We performed brain perfusion measurements from offspring (five days postnatal, P5) of control pregnant dams (sham, n = 13) and of RUPP dams (RUPP, n = 7) using the Pericam® PSI-HR system at a basal condition and after thermal stimuli (warm and cold). We found that pups of RUPP mice exhibited significant differences in perfusion and vascular response to thermal stimuli compared to the sham mice. These differences were associated with high data variability in the Sham group, while in the RUPP group, perfusion looks "stiffer." Data also suggest sex-dimorphism in the vascular response since female pups in the Sham group but not male pups showed statistically significant differences in response to the warm stimulus. Again, this sex-related difference was absent in pups of RUPP mice. In conclusion, we present a robust quantitative approach for LSCI measurements that revealed anomalies in the brain blood flow in offspring of the RUPP model of PE.

Abnormal cerebral microvascular perfusion and reactivity in female offspring of reduced uterine perfusion pressure (RUPP) mice model.

Children born from women with preeclampsia have alterations in cerebral neurovascular development and a high risk for developing cognitive alterations. Because cerebral blood vessels are critical components in cerebrovascular development, we evaluated the brain microvascular perfusion and microvascular reactivity (exposed to external stimuli of warm and cold) in pups born to preeclampsia-like syndrome based on the reduction of uterine perfusion (RUPP). Also, we evaluate the angiogenic proteomic profile in those brains. Pregnant mice showed a reduction in uterine flow after RUPP surgery (-40 to 50%) associated with unfavorable perinatal results compared to sham mice. Furthermore, offspring of the RUPP mice exhibited reduced brain microvascular perfusion at postnatal day 5 (P5) compared with offspring from sham mice. This reduction was preferentially observed in females. Also, brain microvascular reactivity to external stimuli (warm and cold) was reduced in pups of RUPP mice. Furthermore, a differential expression of the angiogenic profile associated with inflammation, extrinsic apoptotic, cancer, and cellular senescence processes as the primary signaling impaired process was found in the brains of RUPP-offspring. Then, offspring (P5) from preeclampsia-like syndrome exhibit impaired brain perfusion and microvascular reactivity, particularly in female mice, associated with differential expression of angiogenic proteins in the brain tissue.

The infinite possibilities of RNA therapeutics.

Although the study of ribonucleic acid (RNA) therapeutics started decades ago, for many years, this field of research was overshadowed by the growing interest in DNA-based therapies. Nowadays, the role of several types of RNA in cell regulation processes and the development of various diseases have been elucidated, and research in RNA therapeutics is back with force. This short literature review aims to present general aspects of many of the molecules currently used in RNA therapeutics, including in vitro transcribed mRNA (IVT mRNA), antisense oligonucleotides (ASOs), aptamers, small interfering RNAs (siRNAs), and microRNAs (miRNAs). In addition, we describe the state of the art of technologies applied for synthetic RNA manufacture and delivery. Likewise, we detail the RNA-based therapies approved by the FDA so far, as well as the ongoing clinical investigations. As a final point, we highlight the current and potential advantages of working on RNA-based therapeutics and how these could lead to a new era of accessible and personalized healthcare.

Preeclampsia and Increased Permeability Over the Blood-Brain Barrier: A Role of Vascular Endothelial Growth Receptor 2.

BACKGROUND: Cerebral complications in preeclampsia are leading causes of maternal mortality worldwide but pathophysiology is largely unknown and a challenge to study. Using an in vitro model of the human blood-brain barrier (BBB), we explored the role of vascular endothelial growth factor receptor 2 (VEGFR2) in preeclampsia. METHODS: The human brain endothelial cell line (hCMEC/D3) cultured on Tranwells insert was exposed (12 hours) to plasma from women with preeclampsia (n = 28), normal pregnancy (n = 28), and nonpregnant (n = 16) controls. Transendothelial electrical resistance (TEER) and permeability to 70 kDa fluorescein isothiocyanate (FITC)-dextran were measured for the assessment of BBB integrity. We explored possible underlying mechanisms, with a focus on the expression of tight junction proteins and phosphorylation of 2 tyrosine residues of VEGFR2, associated with vascular permeability and migration (pY951) and cell proliferation (pY1175). Plasma concentrations of soluble FMS-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF) were also measured. RESULTS: hCMEC/D3 exposed to plasma from women with preeclampsia exhibited reduced TEER and increased permeability to 70 kDa FITC-dextran. These cells upregulated the messenger ribonucleic acid (mRNA) levels of VEGFR2, and pY951-VEGFR2, but reduced pY1175-VEGFR2 (P < 0.05 in all cases). No difference in mRNA expression of tight junction protein was observed between groups. There was no correlation between angiogenic biomarkers and BBB permeability. CONCLUSIONS: We present a promising in vitro model of the BBB in preeclampsia. Selective tyrosine phosphorylation of VEGFR2 may participate in the increased BBB permeability in preeclampsia irrespective of plasma concentrations of angiogenic biomarkers.

Vascular endothelial growth factor and poor prognosis after ischaemic stroke.

BACKGROUND AND PURPOSE: Systemic inflammation conveys information about ischaemic stroke prognosis. Growth factors with neurotrophic and angiogenesis-regulating properties might provide additional information about sequelae. The prognostic performance of circulating vascular endothelial growth factor (VEGF), placental growth factor, interleukin 6 and C-reactive protein measured after acute ischaemic stroke was evaluated. METHODS: Blood samples were collected from n = 45 patients within 24-48 h of acute ischaemic stroke. The primary outcome was death or moderate to severe disability at 6 months (modified Rankin Scale >2). Logistic regression models were used to determine the area under the receiver operating characteristic curve (AUC). Correlation and principal component analyses were performed to examine interrelationships amongst biomarkers. RESULTS: Vascular endothelial growth factor was elevated in ischaemic stroke patients who died or had moderate to severe disability at six months. Correlation analysis revealed interrelationships between VEGF and HbA1c, triglycerides, erythrocyte sedimentation rate and National Institutes of Health Stroke Scale and Rankin scores, whereas principal component analyses identified VEGF as a major loading factor that discriminated good from poor prognosis. There were no significant differences in AUC using each protein individually to identify patients who had modified Rankin Scale score >2 at 6 months (n = 15/41, AUC 0.61-0.74). However, the AUC increased significantly when combining VEGF with interleukin 6 and C-reactive protein compared to the VEGF-only model (AUC 0.92 vs. 0.67, p = 0.02). CONCLUSION: Circulating VEGF was elevated 24-48 h after acute ischaemic stroke and conveyed prognostic information about moderate to severe disability at 6 months.

Are the Cognitive Alterations Present in Children Born From Preeclamptic Pregnancies the Result of Impaired Angiogenesis? Focus on the Potential Role of the VEGF Family.

Evidence from clinical studies has proposed that children born from preeclamptic women have a higher risk of suffering neurological, psychological, or behavioral alterations. However, to date, the mechanisms behind these outcomes are poorly understood. Here, we speculate that the neurodevelopmental alterations in the children of preeclamptic pregnancies result from impaired angiogenesis. The pro-angiogenic factors vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) are key regulators of both vascular and neurological development, and it has been widely demonstrated that umbilical blood of preeclamptic pregnancies contains high levels of soluble VEGF receptor type 1 (sFlt-1), a decoy receptor of VEGF. As a consequence, this anti-angiogenic state could lead to long-lasting neurological outcomes. In this non-systematic review, we propose that alterations in the circulating concentrations of VEGF, PlGF, and sFlt-1 in preeclamptic pregnancies will affect both fetal cerebrovascular function and neurodevelopment, which in turn may cause cognitive alterations in post-natal life.

Endometrial Stem Cells in Farm Animals: Potential Role in Uterine Physiology and Pathology.

The endometrium is an accessible source of mesenchymal stem cells. Most investigations of endometrial mesenchymal stem cells (eMSCs) have been conducted in humans. In animals, particularly in livestock, eMSC research is scarce. Such cells have been described in the bovine, ovine, caprine, porcine, and equine endometrium. Here we provide the state of the art of eMSCs in farm animals with a focus on the bovine species. In bovines, eMSCs have been identified during the phases of the estrous cycle, during which their functionality and the presence of eMSC-specific markers has been shown to change. Moreover, postpartum inflammation related to endometritis affects the presence and functionality of eMSCs, and prostaglandin E2 (PGE2) may be the mediator of such changes. We demonstrated that exposure to PGE2 in vitro modifies the transcriptomic profile of eMSCs, showing its potential role in the fate of stem cell activation, migration, and homing during pathological uterine inflammation in endometritis and in healthy puerperal endometrium. Farm animal research on eMSCs can be of great value in translational research for certain uterine pathologies and for immunomodulation of local responses to pathogens, hormones, and other substances. Further research is necessary in areas such as in vivo location of the niches and their immunomodulatory and anti-infective properties.

Endometritis and In Vitro PGE2 Challenge Modify Properties of Cattle Endometrial Mesenchymal Stem Cells and Their Transcriptomic Profile.

Mesenchymal stem cells (MSCs) were isolated and characterized from postpartum bovine endometrium of animals with subclinical (n = 5) and clinical endometritis (n = 3) and healthy puerperal females (n = 5). Cells isolated displayed mean morphological features of MSCs and underwent osteogenic, chondrogenic, and adipogenic differentiation after induction (healthy and subclinical). Cells from cows with clinical endometritis did not undergo adipogenic differentiation. All cells expressed mRNAs for selected MSC markers. Endometrial MSCs were challenged in vitro with PGE2 at concentrations of 0, 1, 3, and 10 µM, and their global transcriptomic profile was studied. Overall, 1127 genes were differentially expressed between unchallenged cells and cells treated with PGE2 at all concentrations (763 up- and 364 downregulated, fold change > 2, and P < 0.05). The pathways affected the most by the PGE2 challenge were immune response, angiogenesis, and cell proliferation. In conclusion, we demonstrated that healthy puerperal bovine endometrium contains MSCs and that endometritis modifies and limits some functional characteristics of these cells, such as their ability to proceed to adipogenic differentiation. Also, PGE2, an inflammatory mediator of endometritis, modifies the transcriptomic profile of endometrial MSCs. A similar situation may occur during inflammation associated with endometritis, therefore affecting the main properties of endometrial MSCs.

Splitting of IVP bovine blastocyst affects morphology and gene expression of resulting demi-embryos during in vitro culture and in vivo elongation.

Embryo splitting might be used to increase offspring yield and for molecular analysis of embryo competence. How splitting affects developmental potential of embryos is unknown. This research aimed to study the effect of bovine blastocyst splitting on morphological and gene expression homogeneity of demi-embryos and on embryo competence during elongation. Grade I bovine blastocyst produced in vitro were split into halves and distributed in nine groups (3 × 3 setting according to age and stage before splitting; age: days 7-9; stage: early, expanded and hatched blastocysts). Homogeneity and survival rate in vitro after splitting (12 h, days 10 and 13) and the effect of splitting on embryo development at elongation after embryo transfer (day 17) were assessed morphologically and by RT-qPCR. The genes analysed were OCT4, SOX2, NANOG, CDX2, TP1, TKDP1, EOMES, and BAX. Approximately 90% of split embryos had a well conserved defined inner cell mass (ICM), 70% of the halves had similar size with no differences in gene expression 12 h after splitting. Split embryos cultured further conserved normal and comparable morphology at day 10 of development; this situation changes at day 13 when embryo morphology and gene expression differed markedly among demi-embryos. Split and non-split blastocysts were transferred to recipient cows and were recovered at day 17. Fifty per cent of non-split embryos were larger than 100 mm (33% for split embryos). OCT4, SOX2, TP1 and EOMES levels were down-regulated in elongated embryos derived from split blastocysts. In conclusion, splitting day-8 blastocysts yields homogenous demi-embryos in terms of developmental capability and gene expression, but the initiation of the filamentous stage seems to be affected by the splitting.