On-demand release of a selective MMP-13 blocker from an enzyme-responsive injectable hydrogel protects cartilage from degenerative progression in osteoarthritis.

In osteoarthritis (OA), the degradation of cartilage is primarily driven by matrix metalloprotease-13 (MMP-13). Hence, the inhibition of MMP-13 has emerged as an attractive target for OA treatment. Among the various approaches that are being explored for MMP-13 regulation, blocking of the enzyme with specific binding molecules appears to be a more promising strategy for preventing cartilage degeneration. To enhance effectiveness and ensure patient compliance, it is preferable for the binding molecule to exhibit sustained activity when administered directly into the joint. Herein, we present an enzyme-responsive hydrogel that was designed to exhibit on-demand, the sustained release of BI-4394, a potent and highly selective MMP-13 blocker. The stable and compatible hydrogel was prepared using triglycerol monostearate. The efficacy of the hydrogel to prevent cartilage damage was assessed in a rat model of OA induced by anterior cruciate ligament transection (ACLT). The results revealed that in comparison to the rats administrated weekly with intra-articular BI-4394, the hydrogel implanted rats had reduced levels of inflammation and bone erosion. In comparison to untreated control, the cartilage in animals administered with BI-4394/hydrogel exhibited significant levels of collagen-2 and aggrecan along with reduced MMP-13. Overall, this study confirmed the potential of BI-4394 delivery using an enzyme-responsive hydrogel as a promising treatment option to treat the early stages of OA by preventing further cartilage degradation.

Efficient Nitric Oxide Scavenging by Urea-Functionalized Push-Pull Chromophore Modulates NO-Mediated Diseases.

The excess nitric oxide (NO) produced in the body in response to bacterial/proinflammatory stimuli is responsible for several pathological conditions. The current approaches that target the production of excess NO, either through the inhibition of nitric oxide synthase enzyme or its downstream mediators have been clinically unsuccessful. With an aim to regulate the excess NO, urea-functionalized push-pull chromophores containing 1,1,4,4-tetracyanobuta-1,3-dienes (TCBD) or expanded TCBD (eTCBD) were developed as NO scavengers. The NMR mechanistic studies revealed that upon NO binding, these molecules are converted to uncommon stable NONOates. The unique emissive property of Urea-eTCBD enables its application in vitro, as a NO-sensor. Furthermore, the cytocompatible Urea-eTCBD, rapidly inactivated the NO released from LPS-activated cells. The therapeutic efficacy of the molecule in modulating NO-mediated pathological condition was confirmed using a carrageenan-induced inflammatory paw model and a corneal injury model. While the results confirm the advantages of scavenging the excess NO to address a multitude of NO-mediated diseases, the promising sensing and bioactivity of Urea-eTCBD can motivate further exploration of such molecules in allied areas of research.

Estimation of Heritability under Correlated Errors Using the Full-Sib Model.

In plant and animal breeding, sometimes observations are not independently distributed. There may exist a correlated relationship between the observations. In the presence of highly correlated observations, the classical premise of independence between observations is violated. Plant and animal breeders are particularly interested to study the genetic components for different important traits. In general, for estimating heritability, a random component in the model must adhere to specific assumptions, such as random components, including errors, having a normal distribution, and being identically independently distributed. However, in many real-world situations, all of the assumptions are not fulfilled. In this study, correlated error structures are considered errors that are associated to estimate heritability for the full-sib model. The number of immediately preceding observations in an autoregressive series that are used to predict the value at the current observation is defined as the order of the autoregressive models. First-order and second-order autoregressive models i.e., AR(1) and AR(2) error structures, have been considered. In the case of the full-sib model, theoretical derivation of Expected Mean sum square (EMS) considering AR(1) structure has been obtained. A numerical explanation is provided for the derived EMS considering AR(1) structure. The predicted mean squares error (MSE) is obtained after including the AR(1) error structures in the model, and heritability is estimated using the resulting equations. It is noticed that correlated errors have a major influence on heritability estimation. Different correlation patterns, such as AR(1) and AR(2), can be inferred to change heritability estimates and MSE values. To attain better results, several combinations are offered for various scenarios.

A drug-free strategy to combat bacterial infections with magnetic nanoparticles biosynthesized in bacterial pathogens.

The extensive and indiscriminate use of antibiotics in the ongoing COVID-19 pandemic might significantly contribute to the growing number of multiple drug resistant (MDR) bacteria. With the dwindling pipeline of new and effective antibiotics, we might soon end up in a post-antibiotic era, in which even common bacterial infections would be a challenge to control. To prevent this, an antibiotic-free strategy would be highly desirable. Magnetic nanoparticle (MNP)-mediated hyperthermia-induced antimicrobial therapy is an attractive option as it is considered safe for human use. Given that iron and zinc are critical for bacterial virulence, we evaluated the response of multiple pathogenic bacteria to these elements. Treatment with 1 mM iron and zinc precursors resulted in the intracellular biosynthesis of MNPs in multiple Gram-positive and Gram-negative disease-causing bacteria. The superparamagnetic nanoparticles in the treated bacteria/biofilms, generated heat upon exposure to an alternating magnetic field (AMF), which resulted in an increase in the temperature (5-6 °C) of the milieu with a subsequent decrease in bacterial viability. Furthermore, we observed for the first time that virulent bacteria derived from infected samples harbour MNPs, suggesting that the bacteria had biosynthesised the MNPs using the metal ions acquired from the host. AMF treatment of the bacterial isolates from the infected specimens resulted in a strong reduction in viability (3-4 logs) as compared to vancomycin/ciprofloxacin treatment. The therapeutic efficacy of the MNPs to induce bacterial death with AMF alone was confirmed ex vivo using infected tissues. Our proposed antibiotic-free approach for killing bacteria using intracellular MNPs is likely to evolve as a promising strategy to combat a wide range of bacterial infections.

Molecular docking and molecular dynamics to identify collagenase inhibitors as lead compounds to address osteoarthritis.

Osteoarthritis (OA) is a degenerative disease which affects a large number of individuals. Collagenases, which belong to a class of metalloproteases (MMPs), are responsible for the degradation of cartilage manifested in OA. Inhibition of the catalytic domains of these MMPs is one of the important therapeutic strategies proposed for the prevention of OA. The main objective of this work is to evaluate the binding of curcumin and its metabolites with the active sites of collagenases in comparison to standard inhibitors on the basis of our hypothesis that curcumin/metabolites could exhibit an inhibitory effect on MMPs. Here, we report the molecular docking analysis of curcumin and its metabolites with collagenases (MMP-1, MMP-8, MMP-13). Among the molecules tested, curcumin monoglucuronide (CMG) demonstrated the best binding affinity with MMP-13, which is specifically implicated in OA. The CMG-MMP-complexes were further subjected to molecular dynamic simulations to explore the stability of the complexes and to estimate the free binding energies. The results indicated that CMG preferentially bind to MMP-13 in comparison to that of MMP-1 and MMP-8 with binding free energies (ΔGbind) of (-60.55), (-27.02) and (-46.91) kcal/mol, respectively. This is the first study which suggests that curcumin monoglucuronide can be considered as an effective lead compound to prevent the progression of OA.Communicated by Ramaswamy H. Sarma.

COVID-19 associated rhino-orbital-cerebral mucormycosis: An observational study from Eastern India, with special emphasis on neurological spectrum.

AIMS: 1: Describe the epidemiology and determine risk factors for COVID-19 associated mucormycosis. 2: Elaborate the clinical spectrum of Rhino-Orbital-Cerebral Mucormycosis (ROCM), pattern of neuroaxis involvement and it's radiological correlates. METHODS: Observational study. Consecutive, confirmed cases of mucormycosis (N = 55) were included. A case of mucormycosis was defined as one who had clinical and radiological features consistent with mucormycosis along with demonstration of the fungus in tissue via KOH mount/culture/histopathological examination (HPE). Data pertaining to epidemiology, risk factors, clinico-radiological features were analysed using percentage of total cases. RESULTS: Middle aged, diabetic males with recent COVID-19 infection were most affected. New onset upper jaw toothache was a striking observation in several cases. Among neurological manifestations headache, proptosis, vision loss, extraocular movement restriction; cavernous sinus, meningeal and parenchymal involvement were common. Stroke in ROCM followed a definitive pattern with watershed infarction. CONCLUSIONS: New onset upper jaw toothache and loosening of teeth should prompt an immediate search for mucormycosis in backdrop of diabetic patients with recent COVID-19 disease, aiding earlier diagnosis and treatment initiation. Neuroaxis involvement was characterized by a multitude of features pertaining to involvement of optic nerve, extraocular muscles, meninges, brain parenchyma and internal carotid artery.

SARS-CoV-2 and tissue damage: current insights and biomaterial-based therapeutic strategies.

The effect of SARS-CoV-2 infection on humanity has gained worldwide attention and importance due to the rapid transmission, lack of treatment options and high mortality rate of the virus. While scientists across the world are searching for vaccines/drugs that can control the spread of the virus and/or reduce the risks associated with infection, patients infected with SARS-CoV-2 have been reported to have tissue/organ damage. With most tissues/organs having limited regenerative potential, interventions that prevent further damage or facilitate healing would be helpful. In the past few decades, biomaterials have gained prominence in the field of tissue engineering, in view of their major role in the regenerative process. Here we describe the effect of SARS-CoV-2 on multiple tissues/organs, and provide evidence for the positive role of biomaterials in aiding tissue repair. These findings are further extrapolated to explore their prospects as a therapeutic platform to address the tissue/organ damage that is frequently observed during this viral outbreak. This study suggests that the biomaterial-based approach could be an effective strategy for regenerating tissues/organs damaged by SARS-CoV-2.

Recent advances in nanotherapeutic strategies that target nitric oxide pathway for preventing cartilage degeneration.

Nitric oxide (NO) is an important inflammatory mediator involved in the development and progression of osteoarthritis (OA). Increased production of NO in the affected joints promote cartilage damage. As NO synthesis is catalysed by the inducible NO synthase (iNOS) enzyme, iNOS inhibition serves as an attractive therapeutic target to prevent NO release. Despite a number of direct and indirect iNOS inhibitor molecules demonstrating chondro-protective effect, none have reached the clinic. Its limited bioavailability and adverse side effects served as a deterrent for pursuing clinical trials in OA patients. With the advent of nanotechnology, interest in targeting NO for preventing cartilage degeneration has revived. In this article, we discuss the limitations of the existing molecules and provide an insight on recent nanotechnology-based strategies that have been explored for the diagnosis and inhibition of NO in OA. These approaches hold promise in reviving the hitherto under explored potential of targeting NO to address OA.

Actin-binding carbon dots selectively target glioblastoma cells while sparing normal cells.

Curcumin, a pleiotropic signalling molecule from Curcuma longa, is reported to be effective against multiple cancers. Despite its promising effect, curcumin had failed in clinical trials due to its low aqueous solubility, stability and poor bioavailability. While several approaches are being attempted to overcome the limitations, the improved solubility observed with curcumin-derived carbon dots appeared to be a strategy worth exploring. To assess if the carbon dots possess bio-activity similar to curcumin, we synthesized carbon dots (CurCD) from curcumin and ethylenediamine. Unlike curcumin, the as-synthesized curcumin carbon dots exhibited excellent solubility, excitation-dependent emission and photostability. The anti-cancer activity evaluated with glioblastoma cells using the well-established in vitro models indicated its comparable/enhanced activity over curcumin. Besides, the selective affinity of CurCD to the actin filament, indicated it's prospective to serve as a marker of actin filaments. In addition, the non-toxic effects observed in normal cells and fish embryos indicated CurCD was more biocompatible than curcumin. While this work reveals the superior properties of CurCD over curcumin, it provides a new approach to explore other plant derived molecules with similar limitations like curcumin.

Adventitial gene transfer of VEGFR-2 specific VEGF-E chimera induces MCP-1 expression in vascular smooth muscle cells and enhances neointimal formation.

BACKGROUND: The role of vascular endothelial growth factors (VEGFs) in neointimal formation has been controversial. VEGF receptor (R)-2 signaling pathway is crucial in bringing about the effects of VEGFs including vasodilatation, endothelial cell migration and proliferation. In this study we have used an established adventitial gene transfer technique, in vitro studies and a novel VEGF-E/PlGF chimera that binds specifically to VEGFR-2, to investigate the role of VEGFR-2 in neointimal formation. METHODS: Intimal hyperplasia was induced in the carotid arteries of cholesterol fed male New Zealand White rabbits using a silastic collar. Adenoviral vectors encoding VEGF-E chimera (1×10(9) pfu/ml) were transferred to the adventitia of the carotid arteries either alone or together with adenoviruses encoding soluble VEGFR-2 (sVEGFR-2). Adenoviruses encoding LacZ were used as controls. All animals were sacrificed 7 days after the gene transfer. RESULTS: Significant increases in neointimal formation, proliferating cells, inflammatory responses and adventitial angiogenesis were observed in the VEGF-E chimera transduced arteries. The number of medial smooth muscle cells expressing VEGFR-2 was significantly (p<0.001) higher. MCP-1 mRNA levels were significantly (p<0.01) increased in the VEGF-E chimera transduced arteries and transduced rabbit aortic smooth muscle cells (p<0.05). Soluble VEGFR-2 (sVEGFR-2) significantly inhibited VEGF-E chimera induced neointimal formation (p<0.01), cellular proliferation (p<0.01), inflammatory responses (p<0.01) and adventitial angiogenesis (p<0.01). CONCLUSIONS: The results indicate that VEGFR-2 mediated signaling could aggravate neointimal formation and suggest a potential therapeutic role of sVEGFR-2 in inhibiting neointimal formation and adventitial angiogenesis.

VEGF-DdeltaNdeltaC mediated angiogenesis in skeletal muscles of diabetic WHHL rabbits.

BACKGROUND: Arterial occlusive disease is often associated with diabetes mellitus and hypercholesterolaemia which may reduce angiogenic potential of several growth factors. Accordingly, the usefulness of therapeutic angiogenesis in the presence of diabetes and hypercholesterolaemia has remained unclear. We evaluated angiogenic effects of the mature form of vascular endothelial growth factor-D (VEGF-D(deltaNdeltaC)) in skeletal muscles in the presence of severe diabetes and hypercholesterolaemia. METHODS: Intra muscular injections of adenoviruses encoding human VEGF-D(deltaNdeltaC) (AdVEGF-D(deltaNdeltaC)) were given in the hind limbs of a group of diabetic hypercholesterolaemic rabbits and adenoviruses encoding LacZ (AdLacZ) were used as a control. All animals were killed 6 days after the gene transfer. RESULTS: Capillary count, capillary area, capillary permeability and perfusion were significantly higher in the AdVEGF-D(deltaNdeltaC) transduced muscles compared with the AdLacZ controls. Expressions of endothelial nitric oxide synthase (eNOS) and VEGF receptor(R)-2 were also significantly increased in the VEGF-D(deltaNdeltaC) transduced muscles, along with an increased expression of angiopoietins (Angs) and neuropilin-2 (NP-2). Furthermore, VEGF-D(deltaNdeltaC) gene transfer to the skeletal muscles increased localized recruitment of cells with endothelial progenitor-like characteristics. CONCLUSIONS: VEGF-D(deltaNdeltaC) gene transfer can induce efficient angiogenesis in the presence of severe diabetes and hypercholesterolaemia by upregulating eNOS and VEGFR-2 expression. VEGF-D(deltaNdeltaC) appears to be a promising agent for inducing therapeutic angiogenesis even in cases with severe diabetes and hypercholesterolaemia.

Molecular genetics of atherosclerosis.

Atherosclerosis is a complex multifocal arterial disease involving interactions of multiple genetic and environmental factors. Advances in techniques of molecular genetics have revealed that genetic polymorphisms significantly influence susceptibility to atherosclerotic vascular diseases. A large number of candidate genes, genetic polymorphisms and susceptibility loci associated with atherosclerotic diseases have been identified in recent years and their number is rapidly increasing. In this review we focus on some of the major candidate genes and genetic polymorphisms associated with human atherosclerotic vascular diseases.

Gene therapy to prevent occlusion of venous bypass grafts.

Revascularization with vein grafts is standard surgical therapy for occlusive arterial diseases. Autologous saphenous vein grafts are important conduits for repairing blocked coronary arteries and are used in the majority of vein graft procedures. Up to 50% of saphenous vein grafts will be occluded during the first decade after surgery. Vein graft occlusion occurs as a result of neointimal hyperplasia, which takes place in response to hemodynamic changes and vessel wall injury, and is characterized by the migration and proliferation of vascular smooth muscle cells. Intimal hyperplasia is further complicated by the concomitant development of atherosclerosis and thrombosis. In the absence of effective pharmacological interventions for the treatment and prevention of occlusive vein graft disease, gene therapy has emerged as a potential therapeutic alternative. Gene therapy could improve vein graft patency by reducing early thrombosis, neointimal hyperplasia and atherosclerosis. In this review we will summarize the emerging applications of gene therapy as a therapeutic tool in occlusive vein graft disease.

VEGF-A, VEGF-D, VEGF receptor-1, VEGF receptor-2, NF-kappaB, and RAGE in atherosclerotic lesions of diabetic Watanabe heritable hyperlipidemic rabbits.

Plaque angiogenesis may be associated with the development of unstable and vulnerable plaques. Vascular endothelial growth factors (VEGFs) are potent angiogenic factors that can affect plaque neovascularization. Our objective was to determine the effect of diabetes on atherosclerosis and on the expression of angiogenesis-related genes in atherosclerotic lesions. Alloxan was used to induce diabetes in male Watanabe heritable hyperlipidemic (WHHL) rabbits that were sacrificed 2 and 6 months after the induction of diabetes. Nondiabetic WHHL rabbits served as controls. Blood glucose (Glc), serum-free fatty acids (FFA), and serum triglyceride levels were significantly higher in diabetic rabbits. Accelerated atherogenesis was observed in the diabetic WHHL rabbits together with increased intramyocellular lipids (IMCL), as determined by 1H-NMR spectroscopy. Atherosclerotic lesions in the diabetic rabbits had an increased content of macrophages and showed significant increases in immunostainings for vascular endothelial growth factor (VEGF)-A, VEGF-D, VEGF receptor-1, VEGF receptor-2, RAGE, and NF-kappaB. VEGF-A165 and VEGFR-2 mRNA levels were significantly increased in aortas of the diabetic rabbits, where a trend toward increased plaque vascularization was also observed. These results suggest that diabetes accelerates atherogenesis, up-regulates VEGF-A, VEGF-D, and VEGF receptor-2 expression, and increases NF-kappaB, RAGE, and inflammatory responses in atherosclerotic lesions in WHHL rabbits.

Biology of vascular endothelial growth factors.

Angiogenesis is the process by which new blood vessels are formed from existing vessels. The vascular endothelial growth factors (VEGFs) are considered as key molecules in the process of angiogenesis. The VEGF family currently includes VEGF-A, -B, -C, -D, -E, -F and placenta growth factor (PlGF), that bind in a distinct pattern to three structurally related receptor tyrosine kinases, denoted VEGF receptor-1, -2, and -3. VEGF-C and VEGF-D also play a crucial role in the process of lymphangiogenesis. Here, we review the biology of VEGFs and evaluate their role in pathological angiogenesis and lymphangiogenesis.

Adenovirus-mediated gene transfer of placental growth factor to perivascular tissue induces angiogenesis via upregulation of the expression of endogenous vascular endothelial growth factor-A.

Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family that binds specifically to VEGF receptor (VEGFR)-1. However, the mechanism of PlGF- and VEGFR-1-mediated angiogenesis has remained unclear and some in vitro studies suggest that VEGF-A/VEGFR-2 signaling may also play a role in PlGF-mediated angiogenesis. To clarify these issues we evaluated angiogenic responses in a well-characterized periadventitial angiogenesis model using adenovirus-mediated PlGF-2 (AdvPlGF-2) gene transfer. We also investigated the roles of VEGFR-1 and VEGFR-2 in PlGF-2-mediated angiogenesis. Using a periadventitial collar technique, AdvPlGF-2 (1 x 10(9) plaque-forming units/ml) was transferred to the adventitia of New Zealand White rabbits alone or together with adenoviruses encoding soluble VEGFR-1 (sVEGFR-1) or soluble VEGFR-2 (sVEGFR-2). Adenoviruses encoding LacZ were used as controls. All animals were killed 7 days after gene transfer. Increased neo-vessel formation, upregulation of endogenous VEGF-A expression, and a significant inflammatory response were seen in AdvPlGF-2-transduced arteries. The neo-vessels were large and well perfused. sVEGFR-1 and sVEGFR-2 suppressed the angiogenic response of PlGF-2 by 80 and 71.7%, respectively. We conclude that adenovirus-mediated PlGF-2 gene transfer to vascular tissue increases endogenous VEGF-A expression and produces significant angiogenesis. Both sVEGFR-1 and sVEGFR-2 can inhibit PlGF-2-mediated angiogenesis. PlGF-2 is a potentially useful candidate for the induction of therapeutic angiogenesis in vivo.

Gene transfer into rabbit arteries with adeno-associated virus and adenovirus vectors.

BACKGROUND: Gene transfer offers considerable potential for altering vessel wall physiology and intervention in vascular disease. Therefore, there is great interest in developing optimal strategies and vectors for efficient, targeted gene delivery into a vessel wall. METHODS: We studied adeno-associated viruses (AAV; 9 x 10(8) to 4 x 10(9) TU/ml) for their usefulness to transduce rabbit arteries in vivo in comparison with adenoviruses (Adv; 1 x 10(9) to 1 x 10(10) pfu/ml). 100 microl of viruses or placebo solution were injected intraluminally into transiently isolated carotid segments. RESULTS: In normal arteries AAV transduced mainly medial smooth muscle cells (SMC) while Adv transduced exclusively endothelial cells (EC). Mechanical injury to EC layer and internal elastic lamina enabled Adv to penetrate and transduce medial SMC. Transgene expression in EC after the AAV-mediated gene transfer was very low. The use of the EC-specific Tie-1 promoter did not lead to specific transgene expression in EC. Transgene expression in SMC persisted for at least 100 days after the AAV treatment whereas the Adv-mediated effect diminished in 14 days. AAV caused only a modest increase in EC VCAM-1 expression and proliferation rate of vascular cells as compared with the mock-treated arteries while Adv caused an extensive inflammatory cell infiltration, VCAM-1 expression, vascular cell proliferation and morphological damages. CONCLUSIONS: Significant differences were observed between the AAV and the Adv vectors in their patterns of arterial transduction and consequent inflammatory responses. These distinct properties may be utilized for different applications in vascular biology research and gene therapy for cardiovascular diseases.

Gene therapy for cardiovascular diseases.

Gene therapy is a rapidly evolving field of medicine, which potentially offers new treatments for cardiovascular diseases. With the use of gene transfer methods it is possible to modify somatic cells in blood vessels and myocardium to overexpress or inhibit pathologically important proteins and achieve therapeutic effects. Prevention of restenosis after vascular interventions such as percutanous coronary angioplasty (PTCA), percutanous peripheral angioplasty (PTA) or stent implantation, prevention of venous graft failures and therapeutic angiogenesis are the major aims of experimental studies and clinical gene therapy. The promise of gene therapy in the treatment of cardiovascular diseases remains high. Experimental studies have established the proof of principle that gene transfer to cardiovascular system can achieve therapeutic effects. First human clinical trials provided initial evidence of the feasibility and safety of the novel therapy. There are also first successful reports on the prevention of neointimal hyperplasia and promotion of therapeutic angiogenesis in clinical trials. However, there are still important questions regarding utility, efficiency and safety of gene therapy in the treatment of cardiovascular diseases. In this review we discuss the rapid progress in cardiovascular gene therapy, the development of delivery systems and vectors, most promising therapeutic genes and results of the recent human clinical trials.

Angiogenic responses of vascular endothelial growth factors in periadventitial tissue.

Recent discovery of new members of the vascular endothelial growth factor (VEGF) family has generated much interest as to which members may be best suited for therapeutic angiogenesis in various tissues. In this study we evaluated angiogenic responses of the different members of the VEGF family in vivo using adenoviral gene transfer. Adenoviruses (1 x 10(9) plaque-forming units [pfu]) encoding for VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-C(deltaNdeltaC) and VEGF-D(deltaNdeltaC) (deltaNdeltaC are proteolytically cleaved forms) were transferred locally to the periadventitial space of the rabbit carotid arteries using a collar technique that allows efficient local transfection of the periadventitial tissue. Expression of the transfected VEGFs was confirmed by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Seven days after the gene transfer maximum neovessel formation was observed in VEGF-A-, VEGF-D-, and VEGF-D(deltaNdeltaC)-transfected arteries. VEGF-C(deltaNdeltaC) also showed angiogenic activity whereas VEGF-B was not effective in inducing angiogenesis. Pericytes were detected around the neovessels, which also frequently showed the presence of intraluminal erythrocytes. Infiltration of inflammatory cells in response to VEGF-D and VEGF-D(deltaNdeltaC) was less prominent than that caused by other VEGFs. In line with the absence of lymphatics in the normal carotid arteries no significant evidence of lymphatic vessel formation was seen in response to any of the studied VEGFs in the periadventitial space. The results help to define possibilities for local angiogenic therapy around blood vessels and support the concept that angiogenic effects may be tissue-specific and depend both on the growth factor ligands and the target tissues. It is concluded that VEGF-A, VEGF-D, and VEGF-D(deltaNdeltaC) are the best candidates for therapeutic angiogenesis when delivered around large arteries.