Microvascular Growth Research Articles

The Role of Microvascular Variations in the Process of Intervertebral Disk Degeneration and Its Regulatory Mechanisms: A Literature Review.

Microvascular changes are considered key factors in the process of intervertebral disk degeneration (IDD). Microvascular invasion and growth into the nucleus pulposus (NP) and cartilaginous endplates are unfavorable factors that trigger IDD. In contrast, the rich distribution of microvessels in the bony endplates and outer layers of the annulus fibrosus is an important safeguard for the nutrient supply and metabolism of the intervertebral disk (IVD). In particular, the adequate supply of microvessels in the bony endplates is the main source of the nutritional supply for the entire IVD. Microvessels can affect the progression of IDD through a variety of pathways. Many studies have explored the effects of microvessel alterations in the NP, annulus fibrosus, cartilaginous endplates, and bony endplates on the local microenvironment through inflammation, apoptosis, and senescence. Studies also elucidated the important roles of microvessel alterations in the process of IDD, as well as conducted in-depth explorations of cytokines and biologics that can inhibit or promote the ingrowth of microvessels. Therefore, the present manuscript reviews the published literature on the effects of microvascular changes on IVD to summarize the roles of microvessels in IVD and elaborate on the mechanisms of action that promote or inhibit de novo microvessel formation in IVD.

Microvascular rarefaction caused by the NOTCH signaling pathway is a key cause of TKI-apatinib-induced hypertension and cardiac damage.

With the advancement of tumour-targeted therapy technology, the survival of cancer patients has continued to increase, and cardiovascular events have gradually become an important cause of death in cancer patients. This phenomenon occurs due to adverse cardiovascular reactions caused by the cardiovascular toxicity of antitumour therapy. Moreover, the increase in the proportion of elderly patients with cancer and cardiovascular diseases is due to the extension of life expectancy. Hypertension is the most common cardiovascular side effect of small molecule tyrosine kinase inhibitors (TKIs). The increase in blood pressure induced by TKIs and subsequent cardiovascular complications and events affect the survival and quality of life of patients and partly offset the benefits of antitumour therapy. Many studies have confirmed that in the pathogenesis of hypertension, arterioles and capillary thinness are involved in its occurrence and development. Our previous findings showing that apatinib causes microcirculation rarefaction of the superior mesenteric artery and impaired microvascular growth may inspire new therapeutic strategies for treating hypertension. Thus, by restoring microvascular development and branching patterns, total peripheral resistance and blood pressure are reduced. Therefore, exploring the key molecular targets of TKIs that inhibit the expression of angiogenic factors and elucidating the specific molecular mechanism involved are key scientific avenues for effectively promoting endothelial cell angiogenesis and achieving accurate repair of microcirculation injury in hypertension patients.

Vascular co-option in resistance to anti-angiogenic therapy.

Three different mechanisms of neovascularization have been described in tumor growth, including sprouting angiogenesis, intussusceptive microvascular growth and glomeruloid vascular proliferation. Tumors can also grow by means of alternative mechanisms including vascular co-option, vasculogenic mimicry, angiotropism, and recruitment of endothelial precursor cells. Vascular co-option occurs in tumors independently of sprouting angiogenesis and the non-angiogenic cancer cells are described as exploiting pre-existing vessels. Vascular co-option is more frequently observed in tumors of densely vascularized organs, including the brain, lung and liver, and vascular co-option represents one of the main mechanisms involved in metastasis, as occurs in liver and lung, and resistance to anti-angiogenic therapy. The aim of this review article is to analyze the role of vascular co-option as mechanism through which tumors develop resistance to anti-angiogenic conventional therapeutic approaches and how blocking co-option can suppress tumor growth.

MDM2 Regulation of HIF Signaling Causes Microvascular Dysfunction in Hypertrophic Cardiomyopathy.

Microvasculature dysfunction is a common finding in pathologic remodeling of the heart and is thought to play an important role in the pathogenesis of hypertrophic cardiomyopathy (HCM), a disease caused by sarcomere gene mutations. We hypothesized that microvascular dysfunction in HCM was secondary to abnormal microvascular growth and could occur independent of ventricular hypertrophy. We used multimodality imaging methods to track the temporality of microvascular dysfunction in HCM mouse models harboring mutations in the sarcomere genes Mybpc3 (cardiac myosin binding protein C3) or Myh6 (myosin heavy chain 6). We performed complementary molecular methods to assess protein quantity, interactions, and post-translational modifications to identify mechanisms regulating this response. We manipulated select molecular pathways in vivo using both genetic and pharmacological methods to validate these mechanisms. We found that microvascular dysfunction in our HCM models occurred secondary to reduced myocardial capillary growth during the early postnatal time period and could occur before the onset of myocardial hypertrophy. We discovered that the E3 ubiquitin protein ligase MDM2 (murine double minute 2) dynamically regulates the protein stability of both HIF1α (hypoxia-inducible factor 1 alpha) and HIF2α (hypoxia-inducible factor 2 alpha)/EPAS1 (endothelial PAS domain protein 1) through canonical and noncanonical mechanisms. The resulting HIF imbalance leads to reduced proangiogenic gene expression during a key period of myocardial capillary growth. Reducing MDM2 protein levels by genetic or pharmacological methods normalized HIF protein levels and prevented the development of microvascular dysfunction in both HCM models. Our results show that sarcomere mutations induce cardiomyocyte MDM2 signaling during the earliest stages of disease, and this leads to long-term changes in the myocardial microenvironment.

Extracellular Matrix Stiffness Regulates Microvascular Stability by Controlling Endothelial Paracrine Signaling to Determine Pericyte Fate.

The differentiation of pericytes into myofibroblasts causes microvascular degeneration, ECM (extracellular matrix) accumulation, and tissue stiffening, characteristics of fibrotic diseases. It is unclear how pericyte-myofibroblast differentiation is regulated in the microvascular environment. Our previous study established a novel 2-dimensional platform for coculturing microvascular endothelial cells (ECs) and pericytes derived from the same tissue. This study investigated how ECM stiffness regulated microvascular ECs, pericytes, and their interactions. Primary microvessels were cultured in the TGM2D medium (tubular microvascular growth medium on 2-dimensional substrates). Stiff ECM was prepared by incubating ECM solution in regular culture dishes for 1 hour followed by PBS wash. Soft ECM with Young modulus of ≈6 kPa was used unless otherwise noted. Bone grafts were prepared from the rat skull. Immunostaining, RNA sequencing, RT-qPCR (real-time quantitative polymerase chain reaction), Western blotting, and knockdown experiments were performed on the cells. Primary microvascular pericytes differentiated into myofibroblasts (NG2+αSMA+) on stiff ECM, even with the TGFβ (transforming growth factor beta) signaling inhibitor A83-01. Soft ECM and A83-01 cooperatively maintained microvascular stability while inhibiting pericyte-myofibroblast differentiation (NG2+αSMA-/low). We thus defined 2 pericyte subpopulations: primary (NG2+αSMA-/low) and activated (NG2+αSMA+) pericytes. Soft ECM promoted microvascular regeneration and inhibited fibrosis in bone graft transplantation in vivo. As integrins are the major mechanosensor, we performed RT-qPCR screening of integrin family members and found Itgb1 (integrin β1) was the major subunit downregulated by soft ECM and A83-01 treatment. Knocking down Itgb1 suppressed myofibroblast differentiation on stiff ECM. Interestingly, ITGB1 phosphorylation (Y783) was mainly located on microvascular ECs on stiff ECM, which promoted EC secretion of paracrine factors, including CTGF (connective tissue growth factor), to induce pericyte-myofibroblast differentiation. CTGF knockdown or monoclonal antibody treatment partially reduced myofibroblast differentiation, implying the participation of multiple pathways in fibrosis formation. ECM stiffness and TGFβ signaling cooperatively regulate microvascular stability and pericyte-myofibroblast differentiation. Stiff ECM promotes EC ITGB1 phosphorylation (Y783) and CTGF secretion, which induces pericyte-myofibroblast differentiation.

Diabetes diminishes muscle precursor cell-mediated microvascular angiogenesis.

The skeletal muscles of Type II diabetic (T2D) patients can be characterized by a reduced vessel density, corresponding to deficiencies in microvascular angiogenesis. Interestingly, T2D also inhibits the function of many myogenic cells resident within skeletal muscle, including satellite cells, which are well-known for the role they play in maintaining homeostasis. The current study was undertaken to gain a better understanding of the mechanisms whereby satellite cell progeny, muscle precursor cells (MPCs), influence microvascular angiogenesis. Network growth and the expression of genes associated with angiogenesis were reduced when microvessels were treated with conditioned media generated by proliferating MPCs isolated from diabetic, as compared to control rat skeletal muscle, a phenomenon that was also observed when myoblasts from control or diabetic human skeletal muscle were used. When only exosomes derived from diabetic or control MPCs were used to treat microvessels, no differences in microvascular growth were observed. An evaluation of the angiogenesis factors in control and diabetic MPCs revealed differences in Leptin, vascular endothelial growth factor (VEGF), IL1-β, interleukin 10, and IP-10, and an evaluation of the MPC secretome revealed differences in interleukin 6, MCP-1, VEGF, and interleukin 4 exist. Angiogenesis was also reduced in tissue-engineered skeletal muscles (TE-SkM) containing microvessels when they were generated from MPCs isolated from diabetic as compared to control skeletal muscle. Lastly, the secretome of injured control, but not diabetic, TE-SkM was able to increase VEGF and increase microvascular angiogenesis. This comprehensive analysis of the interaction between MPCs and microvessels in the context of diabetes points to an area for alleviating the deleterious effects of diabetes on skeletal muscle.

Vascular Growth in Lymphomas: Angiogenesis and Alternative Ways

The formation of new blood vessels is a critical process for tumor growth and may be achieved through different mechanisms. Angiogenesis represents the first described and most studied mode of vessel formation, but tumors may also use alternative ways to secure blood supply and eventually acquire resistance to anti-angiogenic treatments. These non-angiogenic mechanisms have been described more recently, including intussusceptive microvascular growth (IMG), vascular co-option, and vasculogenic mimicry. Like solid tumors, angiogenic and non-angiogenic pathways in lymphomas play a fundamental role in tumor growth and progression. In view of the relevant prognostic and therapeutic implications, a comprehensive understanding of these mechanisms is of paramount importance for improving the efficacy of treatment in patients with lymphoma. In this review, we summarize the current knowledge on angiogenic and non-angiogenic mechanisms involved in the formation of new blood vessels in Hodgkin’s and non-Hodgkin’s lymphomas.

Angiogenic genes and proteins are differentially expressed in left and right ventricular tissue in perinatal sheep hearts

Birth places a great stress on the fetal heart due to increased cardiac workload, following a disconnection from the low-resistance placenta and closure of fetal shunts. The mechanisms that regulate myocardial microvascular growth and development during the perinatal period are largely unknown. We hypothesized that birth stimulates an increase in angiogenic gene and protein expression in a ventricular dependent manner to reflect the changing roles for the left and right ventricles (LV, RV). Fetal (135 d gestation, dGA; term=147 dGA) and neonatal (1 and 5 postnatal days, PD) sheep LV and RV were snap frozen. Gene expression of key angiogenic regulators ( VEGFA, VEGFR1, VEGFR2, ANGPT1, ANGPT2, PGC1A), endothelial cell growth ( DDL4, NOTCH4, SPP1, PECAM1, NCL), nitric oxide synthesis ( NOS2, NOS3), and extracellular matrix (ECM) remodeling ( MMP2, MMP9, THBS1) were quantified using real-time qPCR (n=12/age group). Protein expression of VEGFA and VEGF-R2 were measured by Western blot (n=5/age group). Data were analyzed by two-way ANOVA with Tukey’s post-hoc test to determine effects of age, ventricle, and interaction. Significance was set at P<0.05. At 135 dGA, all angiogenic, nitric oxide synthesis, ECM, DDL4, SPP1, and PECAM1 mRNA expression levels were higher in the RV vs. LV. At PD1, there were no differences between LV and RV. At PD5, VEGFA, VEGFR1, ANGPT1, ANGPT2, PGC1A, DDL4, SPP1, PECAM1, NCL, MMP2, and THBS1 mRNA expression levels were again higher in the RV vs. LV. At 135 dGA, VEGFA and VEGF-R2 protein expression levels were 76% and 72% lower, respectively, in the RV vs. LV. Within each ventricle, age related changes to mRNA expression patterns were unique. In the LV, mRNA expression of NOTCH4 and NCL increased stepwise from 135 dGA to PD5; however, in the RV, both were upregulated at PD1 but returned to fetal levels by PD5. In contrast, all angiogenic, ECM, DDL4, SPP1, PECAM1, and NOS2 genes were downregulated in the RV at PD1 but all returned to fetal levels by PD5. In the LV, VEGF-R2 protein expression was 56% and 73% lower at PD 1 and 5, respectively, vs. 135 dGA. In summary, gene and protein expression patterns involved in angiogenesis and vascular remodeling change over the perinatal period and are differentially expressed in the ventricles. Greater angiogenic gene expression levels in the RV vs. the LV at 135 dGA might be due to the RV serving as the main systemic pump in utero, thus requiring relatively increased microcirculation. However, reduced VEGFA and VEGF-R2 protein levels at 135 dGA contradict this hypothesis; histological studies to determine vascularity are ongoing. Likewise, the subsequent downregulation of angiogenic genes in the RV at PD1 might be due to the RV’s sudden decrease in cardiac output as the LV becomes the main pump. The underlying reasons for why LV angiogenic genes are not significantly upregulated in the early postnatal period, given its new role as the main systemic pump, are yet to be elucidated. NIH R21-HL152112 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Skeletal muscle adaptation to indirect electrical stimulation: divergence between microvascular and metabolic adaptations.

What is the central question of this study? Can we manipulate muscle recruitment to differentially enhance skeletal muscle fatigue resistance? What is the main finding and its importance? Through manipulation of muscle activation patterns, it is possible to promote distinct microvascular growth. Enhancement of fatigue resistance is closely associated with the distribution of the capillaries within the muscle, not necessarily with quantity. Additionally, at the acute stages of remodelling in response to indirect electrical stimulation, the improvement in fatigue resistance appears to be primarily driven by vascular remodelling, with metabolic adaptation of secondary importance. Exercise involves a complex interaction of factors influencing muscle performance, where variations in recruitment pattern (e.g., endurance vs. resistance training) may differentially modulate the local tissue environment (i.e., oxygenation, blood flow, fuel utilization). These exercise stimuli are potent drivers of vascular and metabolic change. However, their relative contribution to adaptive remodelling of skeletal muscle and subsequent performance is unclear. Using implantable devices, indirect electrical stimulation (ES) of locomotor muscles of rat at different pacing frequencies (4, 10 and 40Hz) was used to differentially recruit hindlimb blood flow and modulate fuel utilization. After 7days, ES promoted significant remodelling of microvascular composition, increasing capillary density in the cortex of the tibialis anterior by 73%, 110% and 55% for the 4Hz, 10 and 40Hz groups, respectively. Additionally, there was remodelling of the whole muscle metabolome, including significantly elevated amino acid turnover, with muscle kynurenic acid levels doubled by pacing at 10Hz (P<0.05). Interestingly, the fatigue index of skeletal muscle was only significantly elevated in 10Hz (58% increase) and 40Hz (73% increase) ES groups, apparently linked to improved capillary distribution. These data demonstrate that manipulation of muscle recruitment pattern may be used to differentially expand the capillary network prior to altering the metabolome, emphasising the importance of local capillary supply in promoting exercise tolerance.

VEGF (Vascular Endothelial Growth Factor) Inhibition and Hypertension: Does Microvascular Rarefaction Play a Role?

Drugs acting by inhibition of the angiogenic action of VEGF (vascular endothelial growth factor) have become major instruments in the treatment of cancer. The downside of their favorable effects in cancer treatment is their frequent cardiovascular side effects. The most consistent finding thus far on the cardiovascular side effects of VEGF inhibitors is the high incidence of hypertension. In this short review, we discuss the evidence that hypertension occurring during VEGF inhibitor treatment is caused by microvascular rarefaction. After a review of the role of VEGF in microvascular growth and differentiation, we present evidence from studies in experimental models of hypertension as well as clinical studies on the microvascular network changes during and after VEGF inhibitor treatment.

Coronary conductance in the normal development of sheep during the perinatal period.

Birth is associated with substantial shifts in cardiovascular physiology. Little is known about coronary vascular adaptations during this period. We used fetal and neonatal lambs to measure coronary function at late gestation (92% of term) and shortly after birth (5-6 days postnatal age). In each animal we measured unanesthetized myocardial perfusion and oxygen delivery using a circumflex artery flow probe. We used inflatable occluders and adenosine to determine coronary conductance and flow reserve. In a subset of animals, we used myocardial contrast echocardiography (MCE, anesthetized) to assess its utility as a tool for studying changes in regional myocardial perfusion in normal development. Separate age-matched animals were instrumented with aortic and coronary sinus sampling catheters to determine myocardial oxygen extraction (unanesthetized). With an average of 17 days of developmental time separating our neonatal and fetal cohorts we found that heart-to-body weight ratio was significantly greater in neonates than fetuses. In resting animals, we found significant decreases in weight-normalized perfusion of, and oxygen delivery to, neonatal relative to fetal myocardium. Similar results were seen when measuring baseline MCE-derived perfusion. Pressure-flow relationship studies revealed lower baseline and maximal coronary conductance in neonates than fetuses, with similar coronary flow reserve between groups. There was greater oxygen extraction in neonates than fetuses. Combined analysis of oxygen extraction with coronary flow suggested greater oxygen consumption by the fetal than neonatal myocardium. We conclude that, during the immediate perinatal period, cardiac growth outpaces coronary microvascular growth resulting in lower capacity for microvascular perfusion in the early neonate.

Bronchopulmonary Dysplasia and Impaired Neurodevelopment-What May Be the Missing Link?

Bronchopulmonary dysplasia (BPD) and poor neurodevelopmental outcome after preterm birth are closely associated. However, mechanistic links are uncertain. We are exploring the hypothesis that decreased circulating insulin-like growth factor (IGF)-1 after preterm birth due to the abrupt end of supply by the placenta impairs growth during critical windows of development in most organs, including the lung and brain. Throughout gestation, the fetus uses glycolysis as its main source of energy. Metabolism is mainly stopped at pyruvate, which serves as a "metabolic crossroad", allowing for the production of amino acids and other "building blocks" for new cells. Metabolic pathways are differentially regulated in the nucleus and the cytoplasm. The ratio between pyruvate dehydrogenase (PDH) and pyruvate dehydrogenase kinase (PDK) determines the biochemical activity which irreversibly metabolizes pyruvate to acetyl-co-A. Metabolites in the nucleus modulate epigenetic remodeling, an essential mechanism of normal growth and maturation during development. IGF-1 has been shown to contribute significantly to the development of virtually all organs, especially related to the regulation of microvascular growth, based on extensive studies of the brain, retina, lung, and intestine. With a preterm birth, the abrupt withdrawal of the placental supply of IGF-1 and its local production directly affects metabolism and microvascular development, which may contribute to a high risk for organ maldevelopment and injury after birth. We speculate that reduced bioavailability of IGF-1 is a possible link between lung and brain development disruption and increases susceptibility for major pulmonary and neurocognitive morbidities in preterm babies. KEY POINTS: · Metabolic changes inherent to prterm birth may cause epigenetic changes which cause "dysmaturational" development.. · IGF-1 may be the potential link between BPD and brain development.. · The ratio between pyruvate dehydrogenase and pyruvate dehydrogenase kinase determines the biochemical activity..

SARS-CoV-2 and Endothelial Cells: Vascular Changes, Intussusceptive Microvascular Growth and Novel Therapeutic Windows.

Endothelial activation in infectious diseases plays a crucial role in understanding and predicting the outcomes and future treatments of several clinical conditions. COVID-19 is no exception. Moving from basic principles to novel approaches, an evolving view of endothelial activation provides insights into a better knowledge of the upstream actors in COVID-19 as a crucial future direction for managing SARS-CoV-2 and other infections. Assessing the function of resting and damaged endothelial cells in infection, particularly in COVID-19, five critical processes emerged controlling thrombo-resistance: vascular integrity, blood flow regulation, immune cell trafficking, angiogenesis and intussusceptive microvascular growth. Endothelial cell injury is associated with thrombosis, increased vessel contraction and a crucial phenomenon identified as intussusceptive microvascular growth, an unprecedented event of vessel splitting into two lumens through the integration of circulating pro-angiogenic cells. An essential awareness of endothelial cells and their phenotypic changes in COVID-19 inflammation is pivotal to understanding the vascular biology of infections and may offer crucial new therapeutic windows.

A modified method of noninvasive computed tomography derived fractional flow reserve based on the microvascular growth space

ObjectiveTo establish a modified method for optimizing outlet boundary conditions (BC) of computed tomography-derived fractional flow reserve (CT-FFR), considering the myocardium as a growth space for microcirculation. The feasibility and diagnostic performance of the modified method in stable coronary artery disease (CAD) were compared with invasive fractional flow reserve (FFR). MethodsNineteen patients (19 lesions) underwent coronary computed tomography angiography (CCTA) and following invasive FFR were included. The microcirculation resistance model generated based on patient-specific anatomical structures and physiological principles was used as the outlet BC, considering the myocardium as a growth space. Brachial artery pressure (BAP) plus or minus 10 mmHg was used as the inlet pressure BC to investigate the effect of the circadian rhythm. After simulation, CT-FFR was compared with invasive FFR with a threshold of 0.80. ResultsCompared with invasive FFR, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of CT-FFR with an optimal threshold of 0.80 were 100%, 100%, 100%, 100%, 100%, respectively. There were a good correlation and consistency between CT-FFR and invasive FFR. Little effect of the circadian fluctuation of BAP was found on the simulation. ConclusionsA modified method for CT-FFR with high diagnostic accuracy compared with invasive FFR was established, considering the whole myocardial as the growth space for microcirculation. Circadian fluctuations in BAP could be ignored when it was used as the inlet BC.

Viewing stromal vascular fraction de novo vessel formation and association with host microvasculature using the rat mesentery culture model.

The objective of the study is to demonstrate the innovation and utility of mesenteric tissue culture for discovering the microvascular growth dynamics associated with adipose-derived stromal vascular fraction (SVF) transplantation. Understanding how SVF cells contribute to de novo vessel growth (i.e., neovascularization) and host network angiogenesis motivates the need to make observations at single-cell and network levels within a tissue. Stromal vascular fraction was isolated from the inguinal adipose of adult male Wistar rats, labeled with DiI, and seeded onto adult Wistar rat mesentery tissues. Tissues were then cultured in MEM +10% FBS for 3 days and labeled for BSI-lectin to identify vessels. Alternatively, SVF and tissues from green fluorescent-positive (GFP) Sprague Dawley rats were used to track SVF derived versus host vasculature. Stromal vascular fraction-treated tissues displayed a dramatically increased vascularized area compared to untreated tissues. DiI and GFP+ tracking of SVF identified neovascularization involving initial segment formation, radial outgrowth from central hub-like structures, and connection of segments. Neovascularization was also supported by the formation of segments in previously avascular areas. New segments characteristic of SVF neovessels contained endothelial cells and pericytes. Additionally, a subset of SVF cells displayed the ability to associate with host vessels and the presence of SVF increased host network angiogenesis. The results showcase the use of the rat mesentery culture model as a novel tool for elucidating SVF cell transplant dynamics and highlight the impact of model selection for visualization.

Impact of the Birth Transition on Striated Muscle Function in Normal Development

Considerable changes are expected in the regulation of myocardial and limb skeletal muscle perfusion from the pre‐ to post‐natal condition based on changes in gravitational force, work load, and arterial O2 content. Yet the impact of birth on the growth and function of microvasculature in striated muscle is poorly understood. Our objective was to assess resting and maximal microvascular flow in the cardiac left ventricle (LV; with high workload before and after birth), and biceps femoris (BF; hindlimb muscle with little workload before birth). We hypothesized that resting flow in the fetus exceeds that in the neonate because of low fetal arterial O2 content, but that flow reserve is greater in the neonate in order to support ambulation. We expected this increase in reserve to be greater in skeletal than myocardial muscle.Fetuses (N=8) were instrumented at 130 d gestational age (dGA, birth is at 146 d) and studied exteriorized from the uterus at 135 dGA. Lambs (N=8) were instrumented 2 d after birth and studied at 5 d. Instrumentation included arterial, venous, and left atrial catheters for systemic pressure monitoring, arterial blood sampling, and drug delivery; and an inflatable occluder around the descending thoracic aorta. Myocardial perfusion (Q) in mL/min/g of tissue was assessed by transthoracic contrast echocardiography at rest, during adenosine‐induced hyperemia, and during combined hyperemia and increased perfusion pressure produced by transient constriction of the postductal aorta. Work was determined by the product of heart rate and perfusion pressure. Hindlimb BF muscle Q was assessed at rest, and during contractile work produced by electrostimulation (2 Hz, 10 mA). Differences were assessed by the Mann‐Whitney U‐test. One fetus and one lamb were excluded from analysis because of abnormal blood gasses at the time of study.At rest, LV myocardial Q was 2‐fold higher in fetuses than neonates (Fig 1A), as was Q normalized to work; whereas, O2 delivery was similar between groups (Fig 1B). Maximal flow during hyperemia followed a similar pattern, with fetal Q, as well as Q per work, exceeding that in the neonate by a factor of 7, whereas maximal O2 delivery per work was similar between age groups. Skeletal muscle (BF) flow in the fetus was highly variable both at rest and during exercise. Median resting Q, resting O2 delivery, and exercised flow were all at least 5‐fold higher in the fetus than neonate, however the difference only reached statistical significance for resting flow (Fig 1A,B). Exercise O2 delivery was similar between ages. Flow reserve was similar between the fetus and neonate in both myocardium and skeletal muscle (Fig 1C).At rest, elevated flow in the fetal LV supported similar levels of O2 delivery per work as found in the newborn. Surprisingly, there was a tendency for increased O2 delivery in the fetal skeletal muscle compared to the newborn; we speculate that this reflects differences in regulation of non‐nutritive flow between the ages studied. Overall, the results of this study suggest that despite the dramatically different physiological circumstances between the fetus and newborn, microvascular growth is regulated to preserve similar levels of flow reserve in both.

Endothelial Regulation of Microvascular Growth and Stability by Ang‐Tie and VEGF Signaling Pathways: A Mechanistic Computational Systems Biology Model

The Angiopoietin‐Tie (Ang‐Tie) pathway is a key signaling pathway regulating vascular stability and permeability, and it significantly intersects and crosstalk with the vascular endothelial growth factor (VEGF) signaling pathway, a major signaling pathway regulating angiogenesis and vascular permeability. Disrupted Ang‐Tie and VEGF signaling is linked to vascular dysfunction related to cancer, systemic inflammation, cardiovascular disease, and diabetic macular edema. Ang‐Tie pathway is regulated by various molecular mechanisms, including ligand‐receptor interactions, receptor multimerization, binding of co‐receptor Tie1, inhibition by vascular endothelial protein tyrosine phosphatase (VE‐PTP), receptor extracellular domain shedding, junctional localization, trafficking, and turnover. VEGF pathway is regulated by ligand binding, homo‐ and heterodimerization of VEGF receptors VEGFR1 and VEGFR2, interaction with neuropilin, thrombospondin‐1/CD47, receptor internalization, recycling, and degradation. Ang‐Tie and VEGF signaling pathways also share crosstalk mechanisms and integrative downstream signaling through phosphoinositide 3‐kinases (PI3K)/Akt, sphingosine‐dependent Erk activation, Src sequestration via RhoA/mDia, and calcium cycling, forming a complex reaction network that requires an integrative model to study the signaling outcomes on a systems level. The present study uses a mechanistic computational model of the Ang‐Tie and VEGF signaling pathways, their regulatory mechanisms, crosstalk, and integrative downstream signaling to quantitatively characterize the molecular control of vascular growth, permeability, and stability by endothelial cells. The model captures and reproduces key experimental observations of the Ang‐Tie/VEGF signaling network with detailed molecular mechanisms. The model significantly expands our previous computational models of the Ang‐Tie signaling pathway [1,2] and integrates our systems biology understanding of the VEGF pathway and their crosstalk. The model serves as a platform for quantitatively predicting the signaling outcome of varying ligand concentrations and receptor expressions in physiological and pathological conditions, identifying molecular mechanisms and targets for therapeutic interventions targeting the signaling network, and simulating the effects of drugs and their combinations on the signaling outcome on a network level.

A Novel ex vivo Method for Investigating Vascularization of Transplanted Islets

Revascularization of transplanted pancreatic islets is critical for survival and treatment of type 1 diabetes. Questions concerning how islets influence local microvascular networks and how networks form connections with islets remain understudied and motivate the need for new models that mimic the complexity of real tissue. Recently, our laboratory established the rat mesentery culture model as a tool to investigate cell dynamics involved in microvascular growth. An advantage is the ability to observe blood vessels, lymphatics, and immune cells. The objective of this study was to establish the rat mesentery tissue culture model as a useful tool to investigate islet tissue integration. DiI-labeled islets were seeded onto adult rat mesentery tissues and cultured for up to 3 days. Live lectin labeling enabled time-lapse observation of vessel growth. During culture, DiI-positive islets remained intact. Radial lectin-positive capillary sprouts with DiI labeling were observed to form from islets and connect to host networks. Lectin-positive vessels from host networks were also seen growing toward islets. PECAM and NG2 labeling confirmed that vessels sprouting from islets contained endothelial cells and pericytes. Our results introduce the rat mesentery culture model as a platform for investigating dynamics associated with the initial revascularization of transplanted islets.

Premarin Reduces Neurodegeneration and Promotes Improvement of Function in an Animal Model of Spinal Cord Injury.

Spinal cord injury (SCI) causes significant mortality and morbidity. Currently, no FDA-approved pharmacotherapy is available for treating SCI. Previously, low doses of estrogen (17β-estradiol, E2) were shown to improve the post-injury outcome in a rat SCI model. However, the range of associated side effects makes advocating its therapeutic use difficult. Therefore, this study aimed at investigating the therapeutic efficacy of Premarin (PRM) in SCI. PRM is an FDA-approved E2 (10%) formulation, which is used for hormone replacement therapy with minimal risk of serious side effects. The effects of PRM on SCI were examined by magnetic resonance imaging, immunofluorescent staining, and western blot analysis in a rat model. SCI animals treated with vehicle alone, PRM, E2 receptor antagonist (ICI), or PRM + ICI were graded in a blinded way for locomotor function by using the Basso–Beattie–Bresnahan (BBB) locomotor scale. PRM treatment for 7 days decreased post-SCI lesion volume and attenuated neuronal cell death, inflammation, and axonal damage. PRM also altered the balance of pro- and anti-apoptotic proteins in favor of cell survival and improved angiogenesis and microvascular growth. Increased expression of estrogen receptors (ERs) ERα and ERβ following PRM treatment and their inhibition by ER inhibitor indicated that the neuroprotection associated with PRM treatment might be E2-receptor mediated. The attenuation of glial activation with decreased inflammation and cell death, and increased angiogenesis by PRM led to improved functional outcome as determined by the BBB locomotor scale. These results suggest that PRM treatment has significant therapeutic implications for the improvement of post-SCI outcome.

Expanding tubular microvessels on stiff substrates with endothelial cells and pericytes from the same adult tissue

Endothelial cells (ECs) usually form a monolayer on two-dimensional (2D) stiff substrates and a tubular structure with soft hydrogels. The coculture models using ECs and pericytes derived from different adult tissues or pluripotent stem cells cannot mimic tissue-specific microvessels due to vascular heterogeneity. Our study established a method for expanding tubular microvessels on 2D stiff substrates with ECs and pericytes from the same adult tissue. We isolated microvessels from adult rat subcutaneous soft connective tissue and cultured them in the custom-made tubular microvascular growth medium on 2D stiff substrates (TGM2D). TGM2D promoted adult microvessel growth for at least 4 weeks and maintained a tubular morphology, contrary to the EC monolayer in the commercial medium EGM2MV. Transcriptomic analysis showed that TGM2D upregulated angiogenesis and vascular morphogenesis while suppressing oxidation and lipid metabolic pathways. Our method can be applied to other organs for expanding organ-specific microvessels for tissue engineering.