The relative concentration of mRNA for each gene was calculated by the Roche LC software utilizing the relative standard curve method. The relative quantity of the gene of interest was normalized by the average relative quantity of four housekeeping genes Gapdh , GusB , Hprt , and 18S. Control and DS samples were averaged, the control samples were set to 1 and the fold-change was calculated.

Western blot analysis calculations were performed in Microsoft Excel. The band intensity was calculated utilizing the Image Lab Software BioRad. The band intensity of the protein of interest was normalized to the band intensity of β-Actin; control samples and DS samples were averaged together, the control samples were set to 1 and the fold-change was calculated.

When compared to controls, postnatal DS lung histology is characterized by diminished alveolarization, defective vascular remodeling and impaired microvascular growth Fig 1. These features are consistent with lungs that characterize the microscopic pathology of bronchopulmonary dysplasia, a neonatal disorder with anti-angiogenic pathobiology [ 26 ].

At high magnification 40x , vascular developmental arrest in DS is characterized by persistence of double capillary layers arrows lining the alveolar spaces, which in contrast, have matured to a single layer in the control lung top.

At medium magnification 20x arterial remodeling defect in DS lung showing a muscular pulmonary artery A with markedly thickened wall while intact remodeling results in thin-walled pulmonary arteries a in the control lung.

DS lung pathologic features are strikingly similar to those of preterm infants with bronchopulmonary dysplasia bottom. Banked human fetal lung tissue with confirmed chromosomal diagnosis of DS was used to test 84 human genes that actively participate in the regulations of angiogenesis.

Most notably, increased expression of the anti-angiogenic genes, COL18A1 endostatin , COL4A3 tumstatin and TIMP3 tissue inhibitor of metallopeptidase 3 mRNA were noted Fig 2 , Table 1.

Out of 84 human genes involved in the regulation of angiogenesis the expression of anti-angiogenic COL18A1 endostatin , COL4A3 tumstatin and TIMP3 Tissue inhibitor of metallopeptidase 3 genes are significantly upregulated as measured by Human Angiogenesis RT2 ProfilerTM PCR Array Qiagen PAHS- Z.

Fetal DS lung samples show increased COL18A , APP and DSCR1 protein expression as measured by Western blot lower panels. While DSCR1 mRNA expression trended towards elevation, statistical significance was noted in COL18A and APP.

In addition to the array studies, individual qPCR and western blot assays were performed to specifically determine whether COL18A1 , APP and DSCR1 mRNA and proteins S1 — S9 Figs are elevated in human fetal DS lungs. Peripheral microvascular density highlighted by CD31 immunostain is significantly decreased in fetal DS lungs when compared to that of control lungs measured by MathLab Image Processing Toolbox Computer Program.

Pulmonary arterial wall thickness in prenatal DS lungs is significantly increased when compared to control lungs measured by MathLab Image Processing Toolbox Computer Program.

In this study we suggest that impaired lung development in Down syndrome DS is caused by increased anti-angiogenic activity during in utero lung development. Utilizing DS fetal lung tissue, we demonstrate diminished microvascular density and increased pulmonary arterial vascular thickness Fig 4 when compared to non-DS controls, suggesting impaired vascular development in DS.

We report elevated lung tissue specific mRNA for anti-angiogenic factors in DS including Collagen18a1 COL18A1 , amyloid protein precursor APP , tumstatin COL4A3 and tissue inhibitor of metallopeptidase 3 TIMP3 Figs 2 and 3.

Elevated lung tissue specific protein for endostatin ES and APP were additionally noted in fetal DS lungs compared to controls Fig 3. Finally, mRNA and protein for Down syndrome critical region 1 DSCR-1 were elevated in DS fetal lungs; significant for protein levels Fig 3.

These findings suggest an in utero anti-angiogenic milieu may contribute to the pulmonary vascular phenotype typical in patients with Down syndrome. The strikingly under-developed pulmonary vascular and alveolar phenotype in patients with Down syndrome DS is similar to the lungs of infants with bronchopulmonary dysplasia Fig 1 and likely contributes to the increased incidence of pulmonary hypertension in this patient population.

These findings are consistent regardless of gestational age, suggesting Down syndrome specific pathomechanisms for disordered lung development. In this study, we are the first to report elevation of anti-angiogenic factors in fetal DS lung tissue including chromosome 21 specific ES, APP, DSCR-1 and non-chromosome 21 specific factors tumstatin and TIMP3.

In utero inhibition of pulmonary vascular growth through these potent inhibitors of VEGF activity likely contributes to the disordered micro-vascular and alveolar growth during critical periods of fetal lung development.

These findings suggest that cardiopulmonary diseases in Down syndrome may primarily be disorders of disrupted vascular development. Prior angiogenesis studies in DS have focused on the beneficial effects of elevated anti-angiogenic factors in preventing vascular lesions and solid tumors [ 17 , 28 ].

Here we emphasize the potential detrimental effects of an elevated anti-angiogenic environment during critical periods of in utero lung development. Although it has been long recognized that infants with DS have a markedly increased risk for developing respiratory disease and severe pulmonary hypertension PAH , underlying mechanisms that contribute to respiratory disease are poorly understood.

Previous reports of elevated serum ES, BAP and tissue DSCR1 [ 18 , 21 , 23 ] have been identified in patients with DS, however the current research emphasis has focused on neoplastic disorders and neurologic dysfunction.

Our findings provide evidence that DS lungs carry features of disrupted pulmonary angiogenesis in fetal life with evidence of defective pulmonary vascular remodeling similar to that seen in patients with pulmonary arterial hypertension.

Of note, a recent study implicated increased circulating serum ES as a potential biomarker to predict adverse outcomes in non-DS adult patients with PAH [ 29 ]. Our findings of elevated ES mRNA and protein in the DS fetal lung may implicate ES and other anti-angiogenic proteins as key factors contributing to the early development of PAH in patients with DS.

It is very likely that overexpressed anti-angiogenic molecules synergistically inhibit DS lung vascular development. For example the function of COL18A1 and COL4A3 are likely symbiotic as COL18A1 blocks VEGF induced endothelial cell migration but not proliferation, while COL4A3 inhibits VEGF induced proliferation and not migration [ 30 ].

In addition to ES and BAP, TIMP3 has been shown to block the VEGF-VEGFR2 binding site further inhibiting VEGF signaling [ 31 ].

Moreover, ES may enhance the anti-angiogenic action of TIMP3 as ES has been shown to inhibit certain matrix metalloproteinases MMP , reducing extracellular matrix degradation and blocking vessel growth [ 32 ].

While TIMP3 and COL4A3 are not expressed on human Chr 21, their mRNA overexpression in fetal DS lungs may be related to an as yet described abnormality in remodeling of extracellular matrix proteins in patients with DS. TIMP3 is known to upregulate matrix metalloproteinase-9 MMP-9 a proteinase that releases tumstatin from the extracellular matrix [ 33 ].

Further investigation may focus on human Chr 21 related TIMP3 modulators of gene or protein expression. In addition to the candidate genes included in this study, there are two additional known potent anti-angiogenic factors on the 21 st human Chr. Over expressed dual-specificity tyrosine- Y -phosphorylation regulated kinase 1A DYRK1A and A disintegrin and metalloproteinase with thrombospondin motifs 1 ADAMTS1 may contribute to the global anti-angiogenic milieu in the DS lung [ 34 , 35 ].

Interestingly, several of these anti-angiogenic factors, in addition to BAP, play a critical role in the development of neurodegenerative disorders that characterize DS [ 35 ]. The individual and synergistic effects of anti-angiogenic factors likely play a significant role in increasing the risk of developing pulmonary vascular disease in patients with DS.

The common comorbid conditions in DS including increased pulmonary vascular hemodynamic stresses from reduced vessel density and structural cardiac defects as well as chronic hypoxemia from airways disease and obstructive sleep apnea likely contribute to the accelerated development of PAH [ 1 , 7 , 8 , 36 , 37 ].

While ES has been directly correlated with PAH disease severity in non-DS adults with PAH [ 29 ], a direct correlation between elevated anti-angiogenic factors and the development of PAH will require further investigation. This novel investigation links the human Chr 21 related anti-angiogenic milieu to the pulmonary vascular phenotype in patients with DS.

While the role of anti-angiogenic factors have been studied with respect to inhibition of tumor growth and neurologic development in this unique patient population, the effect of upregulated anti-angiogenic factors and lung development in patients with DS has never been studied.

This study is limited by its small sample size and likely because of selective tissue degradation we were only able to obtain ES and APP but not BAP protein levels.

Further, ontogeny has not been characterized for COL18A1 , APP and DSCR1 genes and postnatal gene and protein expression into childhood remains unknown, both of which we plan to study in the future.

Identifying the cellular source of the antiangiogenic factors as well as quantification of pulmonary arterial endothelial and smooth muscle cells is of future importance. In summary, utilizing human tissues, we established that potent Chr 21 related anti-angiogenic factors are significantly overexpressed in human DS fetal lung.

By showing that fetal human lung has diminished angiogenesis we speculate that in utero active anti-angiogenic mechanisms significantly contribute to lung hypoplasia and to the increased risk of PAH in DS.

Because of elevated anti-angiogenic gene dosage of triplicated Chr 21, DS has been viewed as a syndrome that carries strong protection against angiogenic diseases. Our data suggest that the pathologic effect of anti-angiogenic function should also be considered in this patient population.

We detected abnormal in utero vessel growth in DS lungs and we propose that this may also take place in other organs including the developing brain. If proven, our findings could serve as basis for translational approaches that focus on early intervention emphasizing angiogenic targets with the goal of reducing pulmonary and neurodegenerative morbidity and mortality in neonates and children with DS.

Conceived and designed the experiments: CG SA. Performed the experiments: CG AM DN BD GS. Analyzed the data: CG AM DB BD GS SA. Wrote the paper: CG AM DB BD SA. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Article Authors Metrics Comments Media Coverage Reader Comments Figures.

Abstract Background and Aims Infants with Down syndrome DS or Trisomy 21, are at high risk for developing pulmonary arterial hypertension PAH , but mechanisms that increase susceptibility are poorly understood.

Methods Human fetal lung tissue from DS and non-DS subjects were obtained from a biorepository. Results The angiogenesis array identified up-regulation of three anti-angiogenic genes: COL18A1 ES , COL4A3 tumstatin and TIMP3 tissue inhibitor of metallopeptidase 3 in DS lungs.

Conclusions We conclude that lung anti-angiogenic factors, including COL18A1 ES , COL4A3 , TIMP3 and APP are over-expressed and fetal lung vessel growth is decreased in subjects with DS. Introduction Down syndrome DS , or Trisomy 21, is associated with significant cardiovascular and pulmonary morbidity and mortality in children, including pulmonary hypertension PAH , chronic hypoxemia, and recurrent respiratory illnesses [ 1 — 4 ].

Materials and Methods Human lung tissue Human lung tissue obtained from the University of Maryland, Baltimore through its NICHD Brain and Tissue Bank for Developmental Disorders NICH Contract HHSNC, Ref.

Quantitative real-time PCR qRT-PCR Total RNA was extracted from human lung tissue using the RNAqueous Total RNA Isolation Kit Life Technologies AM Western Blot Analysis Proteins collected for western blot analysis were collected from whole cell lysates in RIPA buffer Cell Signaling Technology, S with protease Roche, catalog no.

Immunostaining and Morphometric Analysis of Fetal Lung Tissue Histological samples were quantified for vascular density and arterial media wall thickness using the Matlab Image Processing Toolbox Math Works, Inc. Statistics The basic characteristics of each group were compared using an independent two-tailed unpaired t -test using Microsoft Excel software.

Results Abnormal alveolar and vascular structures in DS When compared to controls, postnatal DS lung histology is characterized by diminished alveolarization, defective vascular remodeling and impaired microvascular growth Fig 1. Download: PPT. Fig 1. Expression profiles of angiogenic genes in human fetal lungs with DS Banked human fetal lung tissue with confirmed chromosomal diagnosis of DS was used to test 84 human genes that actively participate in the regulations of angiogenesis.

Fig 2. Anti-angiogenic genes are upregulated in human fetal DS lungs as shown by a volcano plot. Fig 3. COL18A and APP and DSCR1 mRNA expression levels are elevated in human fetal DS lungs as measured by individual qPCR upper panel, left , while array show significant increase in COL18A upper panel, right.

Table 1. Gene expression levels of 84 genes linked to angiogenesis in fetal lung DS tissue compared to that of age-matched controls.

COL18A1 , APP and DSCR1 mRNA and protein are overexpressed in human DS fetal lungs In addition to the array studies, individual qPCR and western blot assays were performed to specifically determine whether COL18A1 , APP and DSCR1 mRNA and proteins S1 — S9 Figs are elevated in human fetal DS lungs.

Discussion In this study we suggest that impaired lung development in Down syndrome DS is caused by increased anti-angiogenic activity during in utero lung development. Supporting Information. S1 Fig. APP protein expression. Western blot gel probed with amyloid protein precursor APP.

s TIF. S2 Fig. Actin protein expression. Western blot APP gel, probed with actin endogenous control. S3 Fig. APP protein blot. White light image of APP western blot. S4 Fig. DSCR1 protein expression. Western blot gel for Down syndrome critical region 1 DSCR1. S5 Fig. Western blot gel, actin control for DSCR1.

S6 Fig. DSCR1 protein blot. White light image of DSCR1 western blot. S7 Fig. ES protein expression. Western blot gel for endostatin ES. S8 Fig. Western blot gel, actin control for ES. S9 Fig. ES protein blot. White light image of endostatin western blot.

Author Contributions Conceived and designed the experiments: CG SA. References 1. McDowell KM, Craven DI. Pulmonary complications of Down syndrome during childhood. The Journal of pediatrics. Salinas M, Elawabdeh N, Lin J, Naguib MM, Hodge MG, Shehata BM.

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DOWNLOAD FOR FREE Share Cite Cite this chapter There are two ways to cite this chapter:. Choose citation style Select style Vancouver APA Harvard IEEE MLA Chicago Copy to clipboard Get citation. Choose citation style Select format Bibtex RIS Download citation. IntechOpen Pulmonary Hypertension Edited by Jean Elwing.

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Introduction The aim of this chapter is to present an overview of salient findings in human beings and animal models particularly in the chicken , as related to known participating molecules in angiogenesis within the lung as a response to induced and natural environmental hypoxia, in the framework of the pathobiology of pulmonary hypertension PH.

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Sands M Howell K Costello C.

To understand mechanisms of vascular remodeling, we examined the effects of Kdr knockout on apoptosis and proliferation of pulmonary vascular cells. At baseline, we observed a mild but significant increase of apoptotic cells Fig. After hypoxic exposure, we found a small but significant increase of PCNA-positive vascular cells within remodeled arterioles Fig.

Conditional Kdr knockout leads to transient apoptosis sustained proliferation and vascular inflammation. After hypoxic exposure, the inflammatory cell ratio was increased, but the combined stimulus of Kdr knockout and hypoxic exposure did not further increase the number of CDpositive cells Fig.

s after Kdr knockout Fig. CO declined after hypoxic exposure, but there was no difference between study and control mice Fig. In line with this, we found no significant differences in cardiac output and left ventricular ejection fraction as measured by cardiac MRT at baseline Fig.

Assessment of pulmonary hypertension and ventricular function by echocardiography and cMRT. White arrows indicate midsystolic notches as a sign of severe PH. To better understand the PH phenotype and the effect of Kdr knockout, we measured BNP and VEGFa.

To investigate if this effect was reproducible in patients under anti-angiogenic therapy, we prospectively investigated baseline and on-treatment VEGFa levels in 34 patients receiving bevacizumab a monoclonal antibody directed against VEGF-a.

In line with our finding in Kdr knockout mice we found a significant increase of serum VEGFa levels on treatment with bevacizumab compared with serum levels before treatment Changes in serum VEGF-a and BNP levels in response to VEGF pathway interruption.

To investigate the effect of anti-angiogenic therapy on the pulmonary vasculature of humans, we investigated lung samples of patients under bevacizumab therapy.

Within the prospective bevacizumab registry of our Oncology department we could identify three patients who underwent pulmonary metastasectomy for colorectal cancer under treatment with bevacizumab. At the time of metastasectomy patients presented in NYHA class I or II.

Computed tomography of the chest prior to metastasectomy illustrated dilated pulmonary arteries Table 2. In all three patients we could identify either islets or vascular obstructive lesions staining positive for VEGFR-3 Fig.

Pulmonary vascular remodeling in colorectal cancer patients on treatment with bevacizumab. Kdr knockout entailed significant down regulation of Cdh5 mRNA Fig.

To investigate whether Kdr knockout leads to increased apoptosis, we investigated C1q , a marker of efferocytosis.

We found C1q mRNA to be significantly upregulated after Kdr knockout, an effect that was attenuated under hypoxic exposure Fig. Gene expression after conditional Kdr deletion. Changes in gene expression were analyzed by ΔΔCt method.

Interestingly Kdr knockout led to a significant decrease of Bmp2 , and Bmpr2 Fig. We found significant downregulation of Bmpr-2 after Kdr knockout, but not after SUGEN treatment Fig.

We investigated the effect of disrupted VEGF signaling on pulmonary vascular disease in a preclinical model of direct ablative gene manipulation of VEGFR We found that endothelial cell-specific knockout in mice leads to a mild PH phenotype that is aggravated by hypoxia.

Moreover we found total vessel occlusion by intimal endothelial cell proliferation and lesions consistent with enMT that resembled the pulmonary arteriopathy of human pulmonary arterial hypertension.

We further hypothesized that anti-angiogenic therapies in cancer patients might cause obstructive pulmonary vascular remodeling. Therefore, we studied plasma and lung specimens from patients treated with bevacizumab, a humanized monoclonal antibody directed against VEGF.

Cardiovascular side-effects of bevacizumab include thromboembolic events [ 22 , 41 ], ischemic events [ 10 , 41 ], hypertension [ 16 , 28 , 55 ], pulmonary embolism [ 22 , 39 , 41 ] and pulmonary hypertension [ 29 ].

The mechanism of these bevacizumab-related cardiovascular events is not fully understood. Adverse effects of VEGF inhibitors are largely consequences of blocking VEGF function in normal vascular physiology including vascular cell turnover and blood pressure regulation [ 21 ].

Preclinical evidence has shown that VEGF blockade leads to endothelial cell apoptosis in most organ systems [ 6 ]. Interestingly, this effect is reversible, resulting in vessel regrowth and normal vessel density after 1—2 weeks [ 31 ].

Histologic evaluation of lung samples obtained from pulmonary metastasectomy of patients on bevacizumab treatment showed similar vascular alterations as seen in our rodent model.

We observed increased media wall thickness, perivascular fibrosis and total vessel occlusions. We assume that intimal hyperplasia may be due to selection of abnormal apoptosis-resistant endothelial cells [ 27 , 37 , 52 ].

Experimental proliferative pulmonary vasculopathy in a rat model was first described by Taraseviciene-Stewart who applied the VEGF receptor blocker SU in combination with hypoxia [ 48 ]. More recently Ciuclan could replicate this model in mice and also observed histological changes resembling those seen in human disease [ 11 ].

Because Sugen systemically suppresses VEGFR-2 VEGFR-1, platelet-derived growth factor receptor, c-Kit stem cell factor receptor and RET tyrosine kinase receptor in all cell types and also causes emphysema [ 25 ], we selectively disrupted only VEGFR-2 signaling in endothelial cells, to dissect this pathway in PH and to overcome the pleiotropic effects on different pulmonary cells including alveolar cells type 1 and 2 [ 25 , 36 , 51 ].

Consequently and in contrast to the Sugen models, we did not observe severe emphysema after Kdr knockout. Therefore, we conclude that emphysema as it was observed in Sugen rat models is unlikely to depend on endothelial cell death alone.

As expected [ 11 , 48 ], we found that mice with disrupted VEGFR-2 signaling develop more extensive PH and RV hypertrophy than wild-type animals exposed to chronic hypoxia. In contrast to Ciuclan, but in line with Taraseviciene-Stewart, we also observed a mild PH phenotype after inhibition of VEGFR signaling without hypoxic exposure.

Most importantly, we found proliferative vascular lesions expressing endothelial cell markers and VEGFR There was no systemic response to Kdr knockout and mean systemic arterial pressure did not change in any of the treatment groups [ 11 , 46 ]. However, because Ciuclan reported a left heart failure phenotype in mice following VEGFR blockade, we investigated the effect of Kdr knockout on LV function utilizing transthoracic echocardiography and cardiac MRT.

After hypoxic exposure we observed a significant decrease of CO in all experimental groups [ 11 ], however, without further decrease by Kdr knockout.

We used MRT to assess RV function and found significantly decreased RV ejection fraction as a consequence of Kdr knockout.

We hypothesize that mechanisms other than increased RV afterload contribute to altered RV function. Bogaard has shown that isolated RV pressure overload by pulmonary artery banding leads to RV hypertrophy but not failure, whereas angioproliferative pulmonary hypertension results in both hypertrophy and RV failure.

Authors hypothesized that structurally altered pulmonary circulation in PAH releases mediators that interfere with adaptive RV responses already maximally challenged to meet the increased mechanical stress [ 9 ]. Therefore, we analyzed both the pulmonary circulation and the ventricles.

Kdr knockout leads to a loss of microvessels, more in the RV than in the LV, and in the lungs with decreased cross-sectional area of pulmonary vessels and subsequent increase in pulmonary arterial pressure [ 19 , 34 ].

Under hypoxia, major vessel obliterative pulmonary vascular lesions are observed in Kdr knockout mice that resemble intimal proliferative lesions of severe human PAH [ 18 , 54 ]. To understand mechanisms of pulmonary vascular remodeling after Kdr knockout we examined the impact on apoptosis and proliferation of pulmonary vascular cells.

Early after Kdr knockout we observed a small but significant increase in caspase 3-positive cells that was followed by a similar significant increase in PCNA-positive cells under hypoxia.

Furthermore, we observed that the angioproliferative lesions in Kdr Δend mice expressed PCNA, suggesting a proliferative phenotype. Kdr knockout was associated with a robust pulmonary vascular inflammatory response with accumulation of inflammatory cells in arterioles of Kdr Δend mice.

Because perivascular inflammatory infiltrates precede vascular remodeling in the development of PAH [ 40 ], a misguided inflammatory response to vascular injury might contribute to the development of pulmonary vasculopathy [ 40 , 47 ].

However, this cellular infiltrate might also be a response to the initial vascular apoptotic processes that are superseded by angioproliferative responses. Therefore, we investigated mRNA levels of C1q, a protein that is crucial for phagocytic removal of apoptotic cells efferocytosis. We found C1q mRNA to be significantly upregulated after Kdr knockout, which may be a signal for efferocytosis deficiency.

Because VEGFR-2 has been shown to be important for macrophage—mediated efferocytosis, efferocytosis deficiency might also drive the vasculopathy observed in the present model [ 23 , 24 , 53 ]. We hypothesize that once efferocytosis is impaired as a consequence of Kdr knock-out, apoptotic cells persist and trigger inflammation and autoimmunity, leading to vascular occlusion and pulmonary hypertension [ 53 ].

In contrast to Ciuclan we found Kdr knockout to directly affect BMP signaling. We found both Bmp2 and Bmpr2 downregulated after the knockout. Although a direct relationship of VEGF and BMP signaling pathways has not been reported, their interaction seems likely.

Reduced expression of Bmp2 and Bmpr2 suggests that both pathways act in parallel and underlines the proliferative nature of the disease resulting in a loss of patent pulmonary microvasculature, and eventually, in a loss of endothelial markers. We could identify elevated VEGFa levels as consequence of VEGFR-2 knockout or bevacizumab therapy.

These findings may be central to the pathogenesis of pulmonary vasculopathy. In those proliferating cells we found a sustained upregulation of VEGFR-3, which might in part account for the pro-proliferative phenotype. VEGFR-3 shares structural similarities to VEGFR-2 and is capable to bind all members of VEGF ligands preferentially VEGF-C and VEGF-D , promoting angiogenesis and lymphangiogenesis [ 3 ].

Because VEGFR-3 is more subjected to regulation by Notch than VEGFR-2, it may be able to rescue neoangiogenesis once VEGFR-2 is blocked [ 8 ].

We hypothesize that VEGFR-3 overexpression serves as a mechanistic explanation for the proliferative vasculopathy seen in the present model, which underpins the 2-hits-theory [ 51 ].

Thus, these data are consistent with the hypothesis that sustained VEGFR-2 inhibition in endothelial cells activates a stem cell—related cell proliferation mechanism that includes VEGFR-3 protein expression [ 1 , 12 ].

Furthermore, we observed similar VEGFRpositive lesions in all cancer patients treated with bevacizumab. Limitations of our work are the lack of a control group for the human studies, the lack transthoracic echocardiograms and the lack of serum samples of metastasectomy patients.

Not all proliferating ECs were positive for CD31 Fig. Later, these lumenless vessels seem to disappear; however, we have no information on the mechanisms underlying the lack of EC markers in the small vessel compartments of lung and heart.

Presumably, vascular changes in patients are not uniform over both lungs, but focally distributed, leading to segmental PH. We propose that interrupted VEGF signaling leads to a pulmonary arteriopathy in rodents.

In humans receiving anti-VEGF treatment, a similar mechanism may be effective. Our findings illustrate the importance of intact VEGF signaling for the maintenance of pulmonary vascular patency.

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To test this possibility, hypoxic mice received an i. Our 3D EC lineage-tracing data indicated that baicalin significantly enhances neovessel formation in VE-cadherin-CreER T2 Rosalsl-tdTomato mice Figure 7, E and F , and Supplemental Videos 9 and The increased SMC thickness, RVSP, and RVH were significantly ameliorated in Hx-PH mice upon baicalin treatment Figure 7, G—K.

Baicalin treatment did not significantly affect the expression of SASP markers Figure 8A but decreased oxidative stress markers, Cdkn1a expression, and the number of ECs with γH2A. X in Hx-PH mice Figure 8, B—F. Increased angiogenic response and ameliorated PH upon PGC-1α activation by baicalin in the Hx-PH mouse model.

B and C Western blot analysis of PGC-1α protein in the lung. E and F 3D EC lineage-tracing experiment. E Representative images showing increased angiogenic responses induced by baicalin.

G Representative 2D images showing α-SMA—stained arterioles. I Representative RVP data. Suppressed Hx-induced cellular senescence of ECs upon PGC-1α activation by baicalin.

B Representative images showing distal pulmonary arteries stained with CD31 and γH2A. C Proportion of γH2A. D and E Western blot analysis of protein carbonyl in the lung. To examine whether the beneficial angiogenic effect of baicalin was mediated specifically through PGC-1α activation in ECs, we administered baicalin to Ppargc1a eKO mice in hypoxic conditions because baicalin could inhibit SMC proliferation in vitro Baicalin treatment did not enhance PGC-1α protein Supplemental Figure 3 , A—C , Vegfa gene expression Supplemental Figure 3D , or angiogenic response Supplemental Figure 3 , F and G , and could not attenuate Hx-PH model in Ppargc1a eKO mice Supplemental Figure 3 , G—K.

Finally, we treated SuHx mice with baicalin to determine whether other protective pathways might be induced that might be different than just induction of VEGF, because PGC-1α has potentially favorable effects on vascular remodeling in PH 17 , Although baicalin treatment enhanced the PGC-1α protein expression Supplemental Figure 4 , A—C , the Vegfa gene was not upregulated Supplemental Figure 4D and the angiogenic response was not induced Supplemental Figure 4 , E and F, and Supplemental Videos 11 and The increased SMC thickness, RVSP, and RVH were not ameliorated in SuHx mice by baicalin treatment Supplemental Figure 4 , G—K.

These findings suggest that baicalin ameliorated Hx-PH through a PGC-1α—VEGF-mediated angiogenic response. Recent developments in 3D imaging combined with tissue-clearing techniques have greatly increased the understanding of human diseases in various fields of medicine, including neurology 11 and nephrology In the present study, we aimed to evaluate pulmonary vascular remodeling in the Hx and SuHx mouse models using our novel 3D-imaging system to explore the underlying mechanisms of PH pathology varying in severity.

An angiogenic response extending to the peripheral lung tissue was markedly observed in Hx, whereas the severer SuHx model showed less angiogenesis, and angiogenesis was associated with a temporal increase in PGC-1α expression. The mouse CabHx model also suppressed angiogenic response and exacerbated PH compared with the Hx-PH as well as SuHx models.

The Ppargc1a eKO mice were devoid of an angiogenic response and exhibited a deterioration of Hx-PH with increased oxidative stress, cellular senescence, and DNA damage. By contrast, PGC-1α activation led to increased angiogenesis and an overall amelioration of Hx-PH phenotypes.

These results suggested that pulmonary endothelial PGC-1α—mediated angiogenesis is essential for adaptive responses to Hx and might represent a potential therapeutic target for PH.

The number of patients with PH is increasing, and combination therapy with pulmonary vasodilators, including prostacyclin pathway agonists, endothelin receptor antagonists, and nitric oxide—cyclic guanosine monophosphate enhancers, has been shown to improve prognosis and quality of life Numerous studies have measured the expression of many related genes and proteins in the context of human PH, including VEGF However, the mechanisms underlying PH development have remained elusive, and targeted therapies specific to PH are still lacking.

We recently developed a novel 3D-imaging system enabling the visualization of the murine pulmonary vasculature. The use of this system revealed a significant proliferation of ECs, together with SMCs, extending to the peripheral lung tissue in the Hx-PH mouse model This response was not detected using conventional 2D histological examination.

With this novel 3D-imaging system, the EC proliferation also was found to be more obvious in Hx-PH than in the severer SuHx model of PH. The angiogenic response patterns reflected the expression of the angiogenesis-related genes Vegfa and Ppargc1a in the acute phase after hypoxic exposure. This newly identified angiogenic response appears to be essential for the adaptation to acute hypoxic exposure and maintenance of EC function but was suppressed after treatment with SU, a VEGFR2 TKI.

However, in the present study, administration of cabozantinib to hypoxic mice exacerbated Hx-PH with suppression of Ppargc1a and Vegfa expression as well as SU These conflicting results should be due to the difference in response to VEGFR TKI between rats and mice, and further investigations are required to confirm that VEGFR2 inhibition itself induces exacerbation of Hx-PH in mice.

Previous reports have indicated that PGC-1α—responsive genes contribute to metabolic processes by regulating the activity of many transcription factors related to mitochondrial biogenesis, inflammation, and metabolic signaling Among them, HIF-independent VEGF signaling is a key pathway.

Vegf gene expression is controlled by PGC-1α via the orphan nuclear receptor ERRα, which plays a crucial role in blood flow recovery after hind limb ischemia The present study demonstrates that PGC-1α is upregulated after hypoxic exposure, which leads to an upregulation of Vegf expression and increased angiogenesis in Hx-PH mice.

PGC-1α also plays important roles in suppressing mitochondrial oxidative stress and dysfunction in vascular cells, which could suppress cellular senescence.

Underlying mechanisms include induction of mitochondrial antioxidant proteins including MnSOD, catalase, Prx5, Prx3, UCP2, TRXR2, and TRX2 26 ; prevention of angiotensin II—mediated JNK activation 27 ; and amelioration of angiotensin II—induced vascular senescence by enabling forkhead box protein O1 FoxO1 -dependent sirtuin 1 SIRT1 transcription In humans as well as in various rodent models of PH 29 , 30 , pulmonary endothelial senescence is considered a cause of inflammation-associated pulmonary endothelial remodeling and dysfunction.

Furthermore, SASP of senescent pulmonary ECs was shown to be associated with increased proliferation in cultured pulmonary vascular SMCs Accordingly, we hypothesized that pulmonary endothelial PGC-1α is a key regulator of cellular senescence that may prevent the progression of PH. We investigated the role of pulmonary endothelial PGC-1α in cellular senescence and demonstrated that pulmonary endothelial PGC-1α attenuated Hx-induced oxidative stress, cellular senescence, and DNA damage.

Upregulation of pulmonary endothelial PGC-1α might, therefore, promote adaptive angiogenic responses by increasing the expression of VEGF as well as reducing endothelial cellular senescence, DNA damage response, and oxidative stress.

Taken together, these mechanisms might suppress the progression of Hx-PH pathology. PGC-1α is regulated at both transcriptional and posttranscriptional levels.

The transcriptional regulation of PGC-1α is mainly mediated by cyclic AMP response element—binding protein CREB 31 , myocyte enhancer factor 2 MEF2 32 , and FoxO1 33 transcription factors. SIRT1 can alter PGC-1α activity by changing its acetylation status 34 , and AMPK regulates both the acetylation status and expression of PGC-1α 34 , In patients and animals with chronic advanced PH, decreased expression and activity of AMPK 36 and PGC-1α 17 have been reported.

Consistently, mice deficient in SMC-specific Creb 37 or Foxo1 38 , EC-specific Mef2c 39 , Ampk 36 , or inducible systemic Sirt1 40 exhibited deterioration of PH.

The activation of FoxO 41 , MEF2 39 , or AMPK 36 attenuates the progression of PH in mouse models. Our findings in Hx-PH mice support these previous results, indicating that therapies aimed at increasing the activation of PGC-1α could attenuate or prevent pulmonary vascular remodeling in PH.

Overall, our results demonstrate that pulmonary endothelial PGC-1α mediates EC protection and angiogenesis in hypoxic mice. These mechanisms might represent a potential avenue for the development of targeted therapies for the prevention and treatment of PH.

Reagents and solutions. Polyethylene glycol mono- p -isooctylphenyl ether Triton X; CAS was purchased from Nacalai Tesque.

Male mice were used for the experiments. All personnel involved in data collection and analysis were masked to the treatment allocation. The mice either were housed under standard normoxic conditions or were continuously housed in a hypoxic chamber 8.

The hypoxic gas mixture was continuously delivered from a nitrogen gas generator MNT Drug administration. SU CAS was purchased from Bio-Techne. SU was suspended using sonication in a mixture of 0. Cabozantinib was purchased from LC Laboratories and suspended using sonication in a mixture of 0.

Baicalin CAS was purchased from Merck KGaA. The mice were injected i. Measurement of right ventricular pressure. The mice were anesthetized with an i.

The mice were orally intubated, and the lungs were ventilated using a mouse ventilator Shinano Manufacturing Co. RVP signals were relayed to pressure amplifiers PCU; AD Instruments and then were continuously sampled using a PowerLab system AD Instruments and recorded on a computer using Chart software AD Instruments.

Heart rate was typically between and bpm under these conditions. If the heart rate fell below bpm, the measurements were excluded from the analysis.

Assessment of right ventricular hypertrophy. The mice were euthanized by cervical dislocation, and their hearts were excised. The atria were removed, and the right ventricle RV was separated from the left ventricle LV and septum S.

The mice were euthanized by cervical dislocation, and the left lungs were harvested for histological and IHC analyses. The Abs used for IHC staining included α-SMA catalog C; Merck KGaA , CD31 catalog DIA; Dianova , PGC-1α catalog NBP; Novus Biologicals , and γH2.

AX catalog ; Cell Signaling Technology. Images were captured using an Olympus BX51 fluorescence microscope. Morphometry was performed on lung sections obtained from randomly chosen animals.

Pulmonary remodeling was assessed using the percent wall thickness of parenchymal pulmonary arteries classified into small arteries terminal bronchioles and arterioles acini or alveolar ducts.

The pulmonary artery diameter was determined using the ImageJ software NIH. CUBIC tissue-clearing protocol and 3D immunofluorescence staining. CUBIC tissue clearing and 3D immunofluorescent staining were performed as previously described 12 , Mice were anesthetized with an i.

For transcardial perfusion, a gauge needle was inserted into the LV through the apex. The postcaval lobes of right lung were excised and continuously immersed in 30 mL of the CUBIC-1 reagent at 37°C with gentle shaking for 1 week for wholemount staining or for 1 day for samples in which ECs were labeled by tdTomato fluorescent proteins.

The reagent was exchanged every day. Images of tissue-cleared, immunofluorescence-labeled, or fluorescent protein—labeled samples were acquired using multiphoton excitation fluorescence microscopy SP8 Leica Microsystems Ltd. The samples were immersed in the CUBIC-2 reagent during image acquisition.

The tissue was excited at wavelengths of and nm using a Ti:sapphire laser Chameleon Vision II; Coherent.

Image processing and 3D reconstruction maximum intensity projection and volume rendering were performed using Leica application suite X Leica Microsystems Ltd. A 3D lineage-tracing experiment of ECs was performed to evaluate 3-dimensionally the proliferation of preexisting ECs during angiogenesis.

VE-cadherin-CreER T2 ; Rosalsl-tdTomato mice were injected i. at 5 weeks of age and started being housed in a hypoxic chamber or drug administration at 8 weeks of age. The lungs were collected at 11 weeks of age, and 3D images were acquired. The angiogenic response was quantitatively evaluated as the angiogenesis index, as previously described The neovessel length and lung circumference were measured using ImageJ software NIH.

Quantification of vessel density. We performed binary conversion of the acquired 3D images and calculated the integral of the area of interest to quantify vascular density in transparent lungs, as previously described Image similarity analysis by feature matching.

We implemented the AKAZE algorithm 13 , which is used for feature detection and description, in Python Evaluating 3D images in their raw form as x — y plain images with z -stacks is difficult; therefore, we adopted a 2.

Before extraction of feature points of the images, we performed grayscale conversion of 3D reconstructed images to improve the feature extraction accuracy, and the image size was uniformly converted to × pixels. The similarity between 2 images was evaluated by automatically extracting and matching image feature points using the AKAZE algorithm.

The degree of similarity between the images was then calculated by comparing the similarity score, which was defined as the mean distance between feature points.

A lower similarity score corresponded to more similar images. RNA isolation and qRT-PCR. The mice were euthanized by cervical dislocation, and their lungs were excised. Total RNA from lung tissues was extracted using an RNeasy Mini Kit Qiagen. Reverse transcription was performed with 1 μg of total RNA, random hexamers, and reverse transcriptase ReverTraAce; TOYOBO.

qRT-PCR was performed using FastStart Essential DNA Green Master Roche in a LightCycler System II Roche. The expression level of each gene was normalized to that of 18s rRNA. The sequences of the PCR primers are listed in Supplemental Table 1.

Western blotting. The extracted proteins from lung tissues were separated using SDS PAGE and electrophoretically transferred to PVDF membranes Millipore. The primary Abs used included PGC-1α catalog NBP; Novus Biologicals and actin catalog MA; Thermo Fisher Scientific. For the detection of protein carbonylation, Protein Carbonyl Western Blot Detection Kit SHIMA Laboratories Co.

Membranes were incubated with 2,4-dinitrophenylhydrazine solution to form the stable derivative of carbonyl groups. The derivatives were detected by anti-dinitrophenyl Ab. Membranes were exposed to HRP-conjugated secondary Abs, and signals were detected using the Pierce ECL Plus Western Blotting Substrate Thermo Fisher Scientific.

Densitometric analysis of proteins on Western blots was performed using ImageJ software NIH and normalized to the indicated internal control proteins. Data availability.

Underlying data for the manuscript can be accessed in the supplemental material. Data are expressed as mean ± SD. All statistical analyses were performed using Statistical Package for the Social Sciences, version 19 IBM , and graphs were made using R, version 4. P values are indicated in individual figure legends.

Study approval. All animal experiments were approved by the Ethics Committee for Animal Experiments of the University of Tokyo and strictly adhered to the guidelines of the University of Tokyo for animal experiments. TF, NT, HH, M Hatano, and IK contributed to the study conception and design.

TF, HH, SI, GN, H Tokiwa, MK, SM, TS, H Takiguchi, TY, and YK contributed to data acquisition. TF, NT, HH, SI, MK, and S Nishimura contributed to the data analysis. TF, NT, S Nomura, KU, M Harada, H Toko, ET, HA, and IK contributed to the data interpretation.

TF, NT, HM, and IK drafted the manuscript. All authors participated in revising it critically for important intellectual content. We are grateful to the laboratory members in the Department of Cardiovascular Medicine, the University of Tokyo, for their valuable technical assistance, especially Asami Ogawa, Yuko Ishiyama, and Kazuhiko Akiba.

We thank Kohei Miyazono University of Tokyo and Hideyuki Beppu for valuable discussion, and S. Paul Oh for providing Tg Alk1 -cre -L1 L1-Cre. to TF. Copyright: © , Fujiwara et al.

This is an open access article published under the terms of the Creative Commons Attribution 4. Reference information: JCI Insight. Go to The Journal of Clinical Investigation. About Editors Consulting Editors For authors Publication ethics Publication alerts by email Transfers Advertising Job board Contact.

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